Histone deacetylase inhibitors (HDIs) are a class of prominent epigenetic drugs that are currently being tested in hundreds of clinical trials against a variety of diseases. A few compounds have already been approved for treating lymphoma or myeloma. HDIs bind to the zinc-containing catalytic domain of the histone deacetylase (HDACs) and they repress the deacetylase enzymatic activity. The broad therapeutic effect of HDIs with seemingly low toxicity is somewhat puzzling when considering that most HDIs lack strict specificity toward any individual HDAC and, even if they do, each individual HDAC has diverse functions under different physiology scenarios. Here, we review recent mechanistic studies using omics approaches, including epigenomics, transcriptomics, proteomics, metabolomics, and chemoproteomics, methods. These omics studies provide non-biased insights into the mechanism of action for HDIs. Human histone deacetylases (HDACs) are categorized into four classes that are based on sequence homology . Class IHDIs block HDAC enzyme activity by binding to the zinc ion in the catalytic site, which blocks substrate access to the site ,12. HDIsHDIs are being tested in over 500 clinical trials for treating many diseases, including cancers, heart diseases, diabetes, inflammatory diseases, and neurological diseases . SAHA anHistones are the classical substrates for HDACs. HDIs are expected to upregulate histone acetylation levels, remodel chromatin accessibility, and upregulate gene expression. This speculation has proved to be an oversimplification by recent studies. Western blot analysis of total histone extracts can detect altered histone acetylation after HDIs treatment. However, the western blot results can be misleading, since different regions of the chromatin are differentially altered by HDIs, especially when considering that only a small fraction of the transcriptome is altered by HDIs in any given condition . ChromatIn the hepatocarcinoma HepG2 cells or colon adenocarcinoma HT-29 cells that were treated with butyrate or TSA, ChIP-chip analysis with antibodies against acetylated histone H3 or H4 (H3ac or H4ac) revealed that, contrary to the expected histone hyperacetylation, the histone acetylation levels were actually reduced at the transcription start sites (TSS) of actively-transcribed genes after HDI treatment. The chromatin regions with reduced acetylation levels were enriched in genes that were downregulated by the HDIs. Although histone hyperacetylation in some TSS was detected, it seemed to be transient. Trimethylation of the lysine 4 on the histone H3 (H3K4me3), a histone marker for the active promoters near TSS, was not altered by HDIs. The global histone hyperacetylation that was detected by western blot in the same study could be driven by the change in the heterochromatin regions, because the hyperacetylated regions were mainly localized in the nuclear periphery. The pan-HDI TSA showed similar H3ac and H4ac changes as the class I/IIa-selective HDI butyrate in this study .In myelogenous leukemia line K562 that was treated with butyrate or SAHA, DNase I-hypersensitive (DHS) sites, as identified by DNase-seq, displayed enhancer-like properties. These regions were associated with increased gene expression and increased binding of transcription factor PU.1. However, the knockdown of PU.1 did not prevent HDIs-induced increase in accessibility and gene upregulation. The effect on the gene expression showed a high degree overlap between the pan-HDI SAHA and the class I/IIa–selective HDI butyrate .The inhibition of HDAC1 and HDAC2 with small molecule ACY1035 increased the H3K27ac levels around transcription start sites, transcription termination sites, and enhancer regions in SupB15 cells, a cell model of B-cell precursor acute lymphoblastic leukemia. MNase-seq revealed that ACY1035 did not change the nucleosomal occupancy at the silent genes around transcription start and termination sites. Instead, it decreased the nucleosomal occupancy at TSSs of the expressed genes. Longtime treatment of ACY1035 (62 h) can cause even more decrease in the nucleosomal occupancies around TSS of the expressed genes. These results, together with other data in the study, suggested that the inhibition of HDAC1,2 could reduce the chromatin occupancy of chromatin remodelers and other factors that are involved in DNA repair .ATAC-seq is another technique looking at chromatin accessibility. In triple-negative breast cancer (TNBC) cells HCC1806 and MDA-MB-231 treated with LBH589, a combined approach of ATAC-seq, DNA methylation BeadChip, and RNA-sequencing was used to profile genome-wide chromatin accessibility, DNA methylation, and gene expression changes . No majoIn a cutaneous T cell lymphoma (CTCL) study, an analysis of human CTCL and control samples revealed chromatin signatures that distinguished the leukemic and non-malignant T cells. SAHA or FK228 are used to treat CTCL, but only a subset (~40%) of CTCL patients respond to HDIs therapy. ATAC-seq revealed that patients with good response to HDIs showed increased DNA accessibility in CTCL cells after HDIs treatment, while patients that were resistant to HDIs did not. Surprisingly, a comparison of HDIs effects across different cell types revealed that HDIs did not open inaccessible sites in CTCL or non-malignant T cells. Rather, HDIs reinforced the existing pattern of DNA accessibility in each given cell type .PI3 kinase, IFNγ, LKB1, TRAIL, and PDGF mediated signaling. Treatment with SAHA or MS275 partially restored histone acetylation levels H2BK5ac, H4K5ac, and H3K27ac in these regions in tumorigenic cells, as revealed by ChIP-string. This could explain the tumor-specificity of the HDIs. H3K27ac ChIP-seq in five melanoma cell lines further showed that cell lines with lower histone acetylation levels were more sensitive to HDIs-induced cytotoxicity. Both the pan-HDI SAHA and the class I–selective HDI MS275 preferentially inhibited the proliferation of tumorigenic cells as compared to the non-tumorigenic cells -d-glucose was used as a tracer to measure the metabolic flux through glycolysis, the pentose phosphate pathway (PPP), and the Krebs cycle. Butyrate and TSA decreased glycolysis, the use of the oxidative branch of the PPP, and pyruvate dehydrogenase contribution. TSA and butyrate induced similar metabolic profiles and induced the differentiation of HT29 cells, which was different from the metabolic effects of acetyl-CoA precursors. When considering that butyrate itself is an endogenous precursor for acetyl-CoA, these data suggest that the metabolic change of butyrate is mainly due to its HDI activity, rather than a precursor of acetyl-CoA. The study also suggests a potential connection between HDIs-induced metabolic changes and cell differentiation -glucose tracer did not identify any prominent changes in cell metabolism after treatment with PXD101 [In cultured pancreatic cancer BxPC3 cells and hepatocellular cancer Hep1 cells, h PXD101 . In ratsh PXD101 . MGCD010In summary, HDIs can alter the metabolism of amino acids, choline, or carbohydrates, which may contribute to their therapeutic or toxic effects in different tissues.Regardless of the downstream epigenomic, transcriptomic, proteomic, or metabolomic changes that HDIs may cause, it is conceivable that HDIs must have physical interactions with their direct targets. Small molecules are notoriously promiscuous, and it is possible that HDIs could bind to many proteins and alter their functionality, in addition to HDACs. Chemoproteomic approaches with chemical probes allow the systemic identification of proteins bound to a small molecule. These methods have been used to profile HDI-interacting proteins in several studies .The HDIs affinity probes were synthesized by conjugating sepharose to analogs of SAHA or class I and class II-selective HDI givinostat. After exposing the probe with protein extracts from the myelogenous leukemia K562 cells, the captured proteins were quantified with mass spectrometry. Competition analysis with HDIs was then performed to reduce the binding affinity of each HDI with the identified binding proteins, generating a dataset composed of 16 HDIs with over 1000 proteins. Immunofluorescence analysis of acetylated histones or tubulin further validated the range of activity for several HDIs, as determined in the chemoproteomic analysis. Class IIa HDACs were not identified in the assay, which is consistent with their low affinity to hydroxamate and lack of catalytic activities due to a missense mutation in the catalytic pocket . The antA similar approach from a separate study with SAHA-coated sepharose beads and HPLC-MS/MS identified 58 proteins as potential SAHA binding proteins in the HeLa cell lysates. 35 proteins were found as HDAC complex components. Gene ontology analysis showed that 23 proteins were metabolic enzymes, four proteins were involved in cell cycle regulation and differentiation, and two proteins were chaperones. A glucose metabolism enzyme ENO-1 was identified as a novel SAHA-binding protein and may be a target of HDIs .Another study used the chemoproteomic approach to explore the mechanism of the 2-aminobenzamide class of HDIs in treating Friedreich’s ataxia (FRDA) and Huntington’s disease. A chemical probe was synthesized by connecting the HDI compound 106 with two structures: one to form a covalent bond with target proteins upon UV crosslink and another to be conjugated with biotin through click chemistry. This active probe and a control probe were incubated with nuclear protein extracts from FRDA patient-derived neural stem cells. After UV crosslink and biotin conjugation, a quantitative proteomic method using multidimensional protein identification technology (MudPIT) was used to identify the proteins that were specifically captured by the probe. Among the 3003 detected proteins, 883 were enriched more than two-fold with the active probe as compared to the control probe. HDAC1, HDAC2, and HDAC3 showed enrichment, which was confirmed by western blot. Gene ontology analysis showed that the active probe-binding proteins were highly enriched in the translation elongation and ribosome-associated pathways. This finding suggests that RNA processing and translational regulation may contribute to HDIs-mediated therapeutic effect in FRDA .In summary, HDIs can interact with a variety of protein and protein complexes, including those that are not directly associated with HDACs. Their profile of interacting proteins tends to be different for different HDIs.Multiple omics approaches have started to shed light on the mechanisms that underlie therapeutic or toxic effects of HDIs, although the mechanism is likely context-dependent and there is no consensus mechanism on the horizon. Although HDIs can upregulate global histone acetylation levels, HDIs do not always cause histone hyperacetylation, especially at promoter regions near gene transcription start sites. HDIs tend to upregulate and downregulate an equal number of genes, although the relative ratio is dependent on the binding kinetics of the HDIs and the timing of the assay. HDIs regulate the expression of genes that are involved in diverse biological pathways, including cell cycle regulation, cell death, metabolism, and stress responses in many cancer cells. The changes in histone acetylation levels at gene promoters tend to correlate with the changes in gene expression levels in most cases, although the gene expression changes are not always associated with phenotypic changes. In addition to epigenomic and transcriptomic profiling, recently developed proteomics, metabolomics, and chemoproteomics approaches have started to provide multifaceted datasets regarding HDIs. Pan-HDIs tend to have distinct effects from class-selective HDIs in general, although overlapping effects were observed under some conditions. The availability of these omics datasets provides opportunities for data mining with artificial intelligence. For example, the correlation of differential responses to HDIs among cancer cell lines with transcriptomic and proteomic effects of HDIs on these cells could shed light on the downstream targets that mediate certain cellular responses to HDIs. Continuous application of these omics measurements and integrated bioinformatics analysis of the datasets shall provide novel mechanistic insights into the HDIs-mediated biological effects.


Takayasu arteritis (TA) is a chronic and nonspecific inflammatory disease mainly affecting the aorta and its major branches, resulting in the stenosis or occlusion of target arteries. Approximately 50% to 60% of patients with TA likely have renal artery stenosis (RAS), which results in refractory hypertension (HTN) and renal dysfunction. Aortorenal bypass with saphenous vein graft (SVG) is the classical procedure to relieve patients’ symptoms. Graft restenosis is the most common complication during long-term follow-up. However, aortorenal vein graft aneurysm (AVGA) is uncommonly reported, and symptomatic or ruptured AVGA that needs reoperation is even rarer. Long-term follow-up results after AVGA reoperation also remain scare. Here, we introduced the long-term result of a symptomatic AVGA under the reoperation of polytetrafluoroethylene (PTFE) graft replacement and provided a literature review of AVGA reoperation after surgical bypass for RAS.An 18-year-old male complained about mild to severe right lumbar pain for 5 days. He underwent right aortorenal bypass with SVG for TA-induced right renal artery stenosis to relieve refractory HTN and renal dysfunction 2 years ago. However, this patient did not proceed with a follow-up after the procedure. Physical examination showed normal vital signs, and an obvious percussion tenderness over the right kidney region was detected. The updated computed tomography angiography (CTA) revealed a right AVGA with a maximum diameter of 26 mm. No restenosis of the proximal and distal anastomoses was detected.The patient was diagnosed to have right aortorenal vein graft aneurysm at the risk of rupture and Takayasu arteritis.The AVGA was resected with a 6 mm PTFE graft replacement. An end-to-side proximal anastomosis to the orifice of the original anastomosis on the abdominal aorta and an end-to-end distal anastomosis to the distal normal renal artery were made.The patient had an uneventful postoperative clinical course and was discharged from the hospital 5 days after the operation. The 4-year updated CTA revealed no restenosis or aneurysmal degeneration of the prosthetic graft.Symptomatic AVGA that needs reoperation is rare. Prosthetic graft replacement is an effective way to eliminate the risk of potential rupture. A 4-year satisfactory result indicative of a prosthetic graft can be the first choice for aortorenal bypass in RAS without active biological inflammation. In 1908, Takayasu, a Japanese ophthalmologist, first described this disease in a young female patient with retinal neovascularization and absent radial pulses.–8 Endovascular therapy is the preferred initial treatment because it is minimally invasive. However, previous studies about endovascular therapy showed inconsistent results regarding long-term patency.–6 In other studies, surgical bypass as an alternative has demonstrated superior patency rates to endovascular intervention. Aortorenal bypass with a saphenous vein graft (SVG) was previously the optimal option.,8 One of the most common complications of surgery is the restenosis and dilation of SVG. However, reports on aortorenal vein graft aneurysm (AVGA) after surgical repair for RAS induced by TA are few, and ruptured AVGA is even rarer, with only 2 published cases without long-term results after reintervention.,10 In this case, we first reported the long-term result of reintervention for AVGA at the risk of rupture and provided a literature review about the treatment of AVGA after surgical bypass for RAS.Indications for revascularization procedures remain unclear because limited research has reported about the prognosis of patients who have TA with renal artery stenosis (RAS) and do not undergo vascular intervention. Revascularization procedures include endovascular therapy with or without stent and surgical repair with an autologous graft or a prosthetic graft.2An 18-year-old male complained about refractory HTN for 2 years. He was under therapy involving 2 different antihypertensive medicines, and his blood pressure varied from 160/110 mm Hg to 150/100 mm Hg. Physical examination showed the following findings: temperature of 36.7°C, heart rate of 73 bpm, respiratory rate of 16 breaths per minute, and blood pressure of 162/90 mm Hg. Abdominal computed tomography angiography (CTA) revealed a severe stenosis lesion (>70%) in the middle segment of the right renal artery with obvious intimal hyperplasia in the stenosis part and slenderness of the left renal artery , and the blocked time for the right renal artery was limited to 20 minutes. The patient had an uneventful postoperative clinical course and was discharged from the hospital 4 days after the operation. One month after the procedure, eGFR increased to 95 ml/minute, and Crea remained normal. The blood pressure was well controlled by only 1 antihypertensive medicine. CTA showed that the right SVG was patent without any restenosis or dilation  graft was interposed. An end-to-side proximal anastomosis to the orifice of the original anastomosis on the abdominal aorta and an end-to-end distal anastomosis to the distal normal renal artery were made Fig. A and B. 3,8,11TA is a chronic and nonspecific inflammatory disease mainly affecting the aorta and its major branches, resulting in the stenosis or occlusion of target arteries. Approximately 50% to 60% of patients with TA likely have RAS, which results in refractory HTN, cardiac inefficiency, and renal dysfunction. The revascularization of the renal artery may be necessary when symptoms worsen. Though revascularization procedures include endovascular therapy with or without stent deployment and surgical repair, studies have reported that surgical revascularization has a long-term patency and a lower risk of restenosis compared with those of endovascular therapy. Otherwise, surgical repair is more suitable for long segments and severe stenosis and have a broader indication than endovascular therapy.–17 In 1973, Stanley first reported the follow-up results of 100 cases of aortorenal SVG, indicating that SVG has a higher likelihood of dilation or aneurysmal degeneration in a pediatric population; a long-term follow-up is necessary for the assessment development of SVG. However, in a review about the complications of renal artery reconstruction that requires reoperation, the aneurysmal deterioration of vein grafts is an uncommon reason, accounting for only 3 (1%) of 278 aortorenal saphenous vein bypass grafts requiring revision. Dean also reported that about 20% (8/39) of the aortorenal SVG dilates, whereas AVGA is detected in only 2 patients without growth during follow-up. In a report on anastomotic aneurysms after the surgical repair of TA, the incidence of anastomotic aneurysm in 103 patients with surgically treated TA is 8.5%. However, aneurysm degeneration involves anastomosis, not saphenous vein bypass. Otherwise, the involved arteries are the aorta, the subclavian artery, and the carotid artery; furthermore, 25% lesions of aneurysms in TA indicate that aneurysmal TA is an independent predictor of the development of an anastomotic aneurysm.The reconstruction of the renal artery with the saphenous vein and a prosthetic material as a bypass graft is durable for atherosclerotic disease. However, the risk of SVG dilation in pediatric patients and patients without atherosclerosis remains unresolved. For the long-term patency of an aortorenal bypass graft, most studies have focused on SVG complications or prosthetic graft restenosis and SVG dilation. Limited studies have described AVGA, especially symptomatic or ruptured AVGA. Rarer cases of AVGA reoperation and long-term results after re-intervention have been reported because most cases of AVGA remain stable during follow-up and are uneventful.,19 Other cases of AVGA after surgical bypass for RAS are either under observation without reoperation or under reoperation without follow-up after discharge. Our case underwent a 4-year follow-up with a satisfactory result. We searched relevant information about reoperations for AVGA after the surgical bypass of RAS via PubMed. The detailed information of the related studies is shown in Table The aneurysm formation of SVG in non-aneurysmal renal artery lesion caused by TA is extremely rare. Cases requiring a reoperation are even rarer, and only 2 cases provided 1- and 2-year follow-up results after the second intervention.–20 We also adopted PTFE graft replacement. Excluding the PTFE graft, Bath reported the replacement of a previous AVGA by using a Dacron graft in 2012. In contrast to our procedure. An alternative autologous graft to SVG has also been proposed. Patterson reported the reconstruction of the renal artery with the radial artery and documented a satisfactory result in a case. Excluding surgical repair for AVGA, Hu also introduced a novel endovascular intervention for AVGA with a cover stent and coils. Berkowitz adopted a new way to prevent the risk of the aneurysmal degeneration of SVG. Our results showed that a tubular SVG supported by an external Dacron mesh could be a suitable graft material for renal reconstruction in a pediatric population. With an average follow-up of 4.3 years, no AVGA was detected in 19 mesh-supported grafts.The most common way to solve AVGA is AVGA replacement by using a prosthetic graft. Five cases of AVGA were successfully resolved by aneurysm resection with an interposed prosthetic graft . However, only 1 case documented a 1-year follow-up with a satisfactory result; 4 other cases did not reveal good results after discharge.Considering the satisfactory result after the 4-year follow-up, we believe that prosthetic graft may be the first choice of graft for patients who have TA-induced RAS and need to undergo aortorenal bypass if no active biological inflammation is detected because biological inflammation increases the likelihood of complications after aortorenal bypass in patients with TA.AVGA after surgical bypass with SVG for TA-induced RAS is uncommon. Symptomatic AVGA requiring a reoperation is even rarer. Prosthetic graft replacement is an effective way to eliminate the risk of potential rupture. However, the long-term result of reoperation for AVGA with prosthetic graft is still scarce. Our case demonstrated a satisfactory 4-year follow-up prosthetic result without restenosis or anastomotic aneurysmal formation, indicating that prosthetic graft might be considered as the first choice for RAS if no active biological inflammation was detected.Conceptualization: Jichun Zhao.Data curation: Bin Huang.Resources: Yi Yang.Software: Yi Yang.Visualization: Ding Yuan.Writing - original draft: Xiyang Chen.Writing - review & editing: Bin Huang, Jichun Zhao.


Patients who undergo upfront curative intent resection for locally advanced squamous cell carcinomas and who have adverse pathologic features benefit from adjuvant therapy. Concurrent cisplatin based chemoradiation is an established standard of care endorsed by national guidelines. Controversy now exists on the applicability of this strategy to the good risk human papilloma virus (HPV) related oropharynx cancer (OPC) patient. Ongoing clinical studies are exploring therapeutic de-escalation in the postoperative setting for this distinct patient population. The introduction of immune checkpoint inhibitors to the therapeutic armamentarium for recurrent/metastatic head and neck cancer patients has led to clinical investigation of incorporation of PD-1 inhibition in the postoperative setting. Therapeutic standards among patients with locally advanced head and neck cancer who have undergone surgical resection have evolved in the past four decades . This isThis review identified landmark prospective clinical trials that provided the foundation for and established current therapeutic standards for postoperative therapy. Pertinent negative trials in the postoperative setting were also included. Ongoing prospective clinical trials in the adjuvant setting were included and cited according to their NCTN identifier. One primary focus is the appropriate postoperative treatment for the prognostically distinct human papilloma virus (HPV) related oropharynx cancer (OPC) with the advent of robotic surgical procedures and the potential for de-escalation in this cohort. Another area of scientific interest involves the incorporation of novel agents to current adjuvant therapy standards, specifically the anti-PD1 inhibitors which are active in the recurrent/metastatic setting , 7. The Definition of high risk featuresThe majority of patients with newly diagnosed mucosal squamous cell carcinomas of the head and neck present with locally advanced disease. A proportion of these patients are candidates for surgical resection. In general, oral cavity primaries are approached with surgical resection if feasible, due to the success of surgical reconstruction in this location. Although organ preserving definitive chemoradiation is well established for squamous cell carcinomas originating from the larynx, certain disease characteristics make upfront surgical resection the preferred therapeutic approach . The recognition of adverse pathologic features after surgical resection have been extensively described in literature that dates back to the 1950's, where factors such as advanced T stage, primary site location, nodal disease burden, and surgical margin involvement were associated with high rates of locoregional failure. Subsequent clinical trials in the 1970's explored postoperative radiation in high risk patients, albeit with significant heterogeneity in the definition of high risk. These studies revealed a locoregional and survival advantage to postoperative radiation . A combiApart from pathologic characteristics, timing of radiation therapy appears to influence the outcome of combined modality treatment. Peters et al.  and Ang 2. Landmark clinical trials in combined modality postoperative therapyRecognition of suboptimal outcomes in high risk patients who receive postoperative adjuvant therapy underscored the need for therapeutic intensification in this patient population. Bachaud et al.  reportedOne meta-analyses of chemotherapy in head and neck cancer (MACH-NC) analyzed the results of 63 prospective studies performed from 1965–1993 . In the p = 0.01). The European Organization for Research and Treatment of Cancer (EORTC) trial 22931 conducted a similar study in which patients with stage III/IV squamous cell cancer of the head and neck were randomized to postoperative chemoradiation with high-dose cisplatin versus radiation alone  R9501 and Southwest Oncology Group (SWOG) 9515 conducted a phase 3 study comparing radiation alone to concurrent chemoradiation with high-dose cisplatin given on days 1, 22, and 43 in patients with squamous-cell carcinoma of the oral cavity, oropharynx, larynx, or hypopharynx who had undergone a complete resection of their disease and had high-risk characteristics (p = 0.04) as was overall survival . Furthermore, the cumulative incidence of local or regional recurrences was significantly lower in the combined-therapy group (p = 0.007). It is notable, however, that grade 3 or higher toxicities were more frequently observed in the group that received combined modality postoperative therapy . The RTOG 9501 intergroup trial and the EORTC trial 22931 played a pivotal role in establishing the current North American guidelines for postoperative combined modality treatment.The maximum radiation dose given was 66 Gy in 33 fractions over a period of 6.5 weeks. After a median follow-up of 5 years, the rate of progression-free survival was significantly higher in the group that received combined modality postoperative therapy , higher rates of disease-free survival , and trends toward an a higher rate of overall survival   .In summary for patients with locally advanced HNSCC who undergo curative intent surgery and have high-risk features , postoperative chemoradiation with cisplatin is the standard of care for those who can tolerate therapy.3. Clinical investigation with alternative chemotherapy or radiation regimensDespite the success of the bolus cisplatin and radiation approach, it is well recognized that further optimization of outcomes in high risk populations is needed. Interest in the incorporation of biological therapy into concurrent chemoradiation seemed an attractive approach to intensifying the systemic therapy component, without the excess toxicity of traditional cytotoxic agents. The epidermal growth factor receptor (EGFr) is nearly universally overexpressed among squamous cell malignancies, and inhibitors appeared to have preclinical synergy with radiation therapy. In the definitive treatment of locally advanced oropharynx, larynx, and hypopharynx cancers, the combination of cetuximab with radiation therapy resulted in superior overall survival, progression free survival, and locoregional control .Harrington et al.  reportedSimilarly, interest in variations of platinum administration is of particular clinical significance as standard bolus cisplatin based chemoradiation results in significant high grade toxicity. For example, the EORTC 22931 study reported that only 61% of the study patients were able to complete all three planned cisplatin doses in patients randomized to chemoradiation. Argiris et al.  conducteFinally, studies of alternative fractionation in locally advanced HNSCC have failed to show a survival benefit , 31. A PHPV-related oropharynx cancerIncreasing recognition of the HPV-related oropharynx squamous cell carcinoma subset has had a tremendous impact on the prospective evaluation of HNSCC. This distinct entity, which carries a superior prognosis both in the locally advanced and the metastatic setting, has led to the design of HPV OPC specific clinical trials and has necessitated stratification for HPV status when studied with non HPV related HNSCC . Given tProspective data is expected from ECOG 3311, a recently completed, randomized, prospective phase II clinical trial for patients with advanced stage HPV associated oropharyngeal squamous cell cancer who have undergone transoral surgery and neck dissection (NCT01898494). In this trial, patients with high-risk features  were assigned to receive standard of care adjuvant chemoradiation therapy. Patients with no high-risk features were assigned to observation ; those with intermediate-risk features  were randomized between standard- (60 Gy/30 fractions) and reduced-dose radiation (50 Gy/25 fractions). The trial's primary objective will be to evaluate 2 years progression free survival (PFS) in HPV-positive HNSCC patients treated with low-dose radiation therapy, while its secondary end points will be early/late toxicities, quality of life, and swallowing function.Two other large postoperative de-escalation Phase III trials are ongoing. PATHOS (NCT02215265) is a multi-institutional randomized trial conducted in the United Kingdom similar to ECOG 3311 that will risk stratify HPV related p16+ HNSCC into low, intermediate or high risk groups. Patients with intermediate risk will be randomized to standard or deescalated postoperative radiation. High risk patients will be randomized to postoperative radiation to 60 Gy or postoperative radiation with weekly cisplatin chemotherapy. ADEPT NCT01687413 trial is a study of postoperative adjuvant therapy de-intensification for HPV-related, p16+ oropharynx cancers. In this trial, HPV-related oropharyngeal cancer patients who have undergone surgery and neck dissection and have been found to have high-risk features are randomized to standard-of-care adjuvant chemoradiation versus radiation alone.In addition, smaller phase II trials are being conducted in North America. The Sinai Robotic Surgery Trial (SIRS - NCT02072148) is a single institution trial which will risk stratify patients to low, intermediate or high risk. Patients with intermediate risk will receive 50 Gy postoperatively, and those with high risk treated with 60 Gy with weekly cisplatin chemotherapy. The Mayo Clinic NCT01932697 is conducting a phase II trial wherein patients with high risk features after resection will be treated with altered fractionation with concurrent docetaxel. The University of Pennsylvania (NCT02159703) has an ongoing clinical trial involving adjuvant radiation to the regional lymph nodes only (sparing the primary sites) in patients with low volume T disease, negative margins and pathologic nodal involvement. Lastly, a large German multi-institutional Phase I study, DELPHI (NCT03396718), is ongoing and is examining deescalated radiation doses in patients with low or intermediate risk pathologic features after resection.It is notable that in the definitive setting, de-intensification studies for HPV-related oropharyngeal cancers have preliminarily shown negative results. RTOG 1016 (NCT01302834) is an ongoing trial comparing radiation therapy with cisplatin or cetuximab in patients with p16 positive oropharyngeal cancers. For now, the question of whether patients with locally advanced HNSCC which are HPV-positive require the same intensity of adjuvant therapy as those which are HPV-negative remains unanswered. Given the preliminary negative studies reported in the definitive setting, it is important to note that de-escalation still has high-risk and should be tested only within the context of a clinical trial. Table 2. Immunotherapy and other therapeutic strategies in the perioperative settingNivolumab and pembrolizumab, two monoclonal antibodies that inhibit PD-1, were approved in 2016 for recurrent/metastatic squamous cell carcinomas of the head and neck previously treated with cisplatin chemotherapy. The encouraging activity of these agents in the metastatic setting gives merit to investigation in the curative intent setting. Preclinical data suggests synergy between the anti-PD-1 inhibitors and radiation therapy, making this approach an attractive one in the high risk postoperative setting .The NRG has recently completed HN003 NCT02775812), a phase I experience in high risk resected HPV related squamous cell carcinomas (defined as positive margin and/or ECE), wherein pembrolizumab 200 mg IV every 3 weeks administered with postoperative radiation and weekly cisplatin chemotherapy. The results of this study are expected to provide a basis for future comparison of this regimen to the current therapeutic standard. Another strategy under study is the administration of the anti-PD1 agents in both the neoadjuvant and adjuvant setting. Wise-Draper et al.  recently775812, aAlongside immunotherapy, a number of other therapeutic strategies are currently under active investigation. The ECOG-ACRIN cancer research group is studying the use of radiation therapy with or without cisplatin in treating patients with p16 negative stage III-IVa HNSCC who have undergone surgery (NCT02734537). This phase II trial mandates central determination of tumor p53 status, and is anticipated to provide valuable information regarding oncologic outcomes based on p53 aberrations, potentially paving the way for genomically driven adjuvant therapy recommendations. Furthermore, translational research is pushing the field toward the development of novel therapeutic strategies, with synthetic lethality proving to be an encouraging avenue for therapy. Increasing preclinical evidence points to the reliance of p53 deficient cells on wee-1, a G2/M checkpoint regulator, to affect DNA repair after exposure to cytotoxic agents. The wee-1 inhibitor—AZD1775—in combination with neoadjuvant weekly docetaxel and cisplatin before definitive therapy in HNSCC had promising findings that may be translated into an innovative therapeutic approach . An ongoWhile concurrent cisplatin based chemoradiation continues to be the standard of care for postoperative management of high risk resected HNSCC, the field is rapidly changing. Improving radiation techniques, checkpoint inhibitors, and novel therapeutic strategies, along with the recognition of HPV status as an important prognostic indicator, may help to increase the probability of cure in patients with advanced head and neck cancers. Ongoing clinical trials will hopefully be able to answer how to rationally combine effective novel therapies in the postoperative setting.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RationaleIn an era of antimicrobial resistance, antimicrobial stewardship programs are tasked with reducing inappropriate use of antimicrobials in community and hospital settings. Intensive care units are unique, high-stakes environments where high usage of broad-spectrum antimicrobials is often seen. Handshake stewardship has emerged as an effective mode of prospective audit and feedback to help optimize antimicrobial usage, emphasizing an in-person approach to providing feedback.ObjectivesSix months following the implementation of handshake stewardship rounds in our intensive care unit, we performed a cross-sectional survey of critical care physicians to assess their attitudes and perceptions towards handshake stewardship rounds and preferred mode of delivery of antimicrobial stewardship prospective audit and feedback strategies.MethodsA web-based survey was distributed to 22 critical care physicians working in our hospital and responses were collected over a two-week period.Measurements and Main ResultsMost critical care physicians believe that handshake stewardship rounds improve the quality of patient care (85.7%) and few believe that handshake stewardship rounds are an ineffective use of their time (14.3%). The majority of critical care physicians believe formal, scheduled rounds with face-to-face verbal interaction are very useful compared to providing written suggestions in the absence of face-to-face interaction (71.4% vs 0%).ConclusionsBased upon our survey results, handshake stewardship is valued amongst the majority of critical care physicians. Antimicrobial stewardship prospective audit and feedback strategies emphasizing face-to-face interaction are favored amongst critical care physicians. Antibiotic stewardship has been defined as the “coordinated interventions designed to improve and measure the appropriate use of antibiotic agents by promoting the selection of the optimal antibiotic drug regimen including dosing, duration of therapy, and route of administration” . Prior sSeveral barriers may impede the success of antimicrobial stewardship interventions in intensive care units, such as diagnostic uncertainty and fear of not adequately treating potential pathogens -16. ProsSix months following the implementation of handshake stewardship rounds occurring three times weekly in our medical-surgical critical care unit, we performed a cross-sectional survey amongst critical care physicians in our center to determine their attitudes and perceptions towards handshake stewardship rounds.A web-based cross-sectional survey was designed and distributed to all critical care physicians working in Saskatoon, Saskatchewan, Canada, six months following the implementation of handshake stewardship rounds in our center.The generation of items for the survey tool was developed in collaboration with experts in infectious diseases, critical care and antimicrobial stewardship. The questions were designed to assess the attitudes and perceptions of antimicrobial stewardship. Survey items assessed attitudes, perceptions and experiences towards handshake stewardship rounds using a five-point Likert-type scale with responses grouped as strongly agree/agree, neutral, and disagree/strongly disagree.The introductory email and link to the online survey were sent in May 2017 using SurveyMonkey  and were distributed to all identified critical care physicians, followed by two reminder emails sent every three days. The introductory email included a description of the purpose of the survey and the value of providing anonymous feedback. Survey responses were collected over a period of two weeks. The research proposal and survey tool were approved by the University of Saskatchewan Research Ethics Board.The survey was distributed to 22 critical care physicians who worked in Saskatoon, Saskatchewan, Canada at the time of the survey, with a response rate of 68.2% (n = 15/22). One respondent reported no longer working in critical care so was excluded from our analysis; all 14 respondents included in our analysis reported participating in handshake stewardship rounds in the prior six months. Most respondents (85.7%) who had participated in handshake stewardship rounds reported that these rounds improve the quality of patient care. In addition, the minority of respondents (14.3%) disagreed that handshake stewardship rounds were an effective use of their time.More than half of our respondents (57.1%) reported that handshake stewardship rounds changed their use of antimicrobials in critical care. Amongst these eight individuals reporting a change in the use of antimicrobials, the most commonly reported perceived changes in antimicrobial prescribing behaviors included using more targeted therapy (100%) and using a shorter duration of therapy (50%). Interestingly, 42.8% of survey respondents reported that handshake stewardship rounds resulted in greater discussion of antimicrobial prescribing during their daily morning bedside rounds. Furthermore, most survey respondents (85.7%) disagreed that handshake stewardship rounds impeded upon their autonomy as a critical care physician.Critical care physicians were also asked to rate the perceived utility of different modes of delivering prospective audit and feedback using a four-point Likert-type scale of very useful, somewhat useful, neutral and not useful. The four modalities presented included formal scheduled rounds with face-to-face interaction, face-to-face interaction but not formally scheduled rounds, written suggestions in practitioner orders without face-to-face interaction, and lastly, written suggestions in patient chart progress notes without face-to-face interaction. Formal scheduled rounds with face-to-face verbal interaction were rated as being very useful and the preferred modality amongst critical care physicians 71.4%), followed by face-to-face verbal interaction outside of formal rounds (42.9%, Figure .4%, follHandshake stewardship rounds are valued and supported amongst most critical care physicians as a form of prospective audit and feedback that improves patient quality of care. In addition, most critical care physicians believe handshake stewardship rounds are an effective use of time and do not impede upon physician autonomy. While many critical care physicians self-reported that handshake stewardship rounds changed their antimicrobial prescribing behaviors and resulted in greater discussion of antimicrobials during their morning rounds, further studies are needed to assess the impact of handshake stewardship rounds on the appropriateness of antimicrobial use and other pertinent clinical outcomes in critical care settings. Our survey supports current literature that the preferred delivery model of prospective audit and feedback amongst critical care physicians involves face-to-face interaction . This reOur survey has several limitations. Firstly, it is possible that our survey may not have captured responses of all critical care physicians who had participated in handshake stewardship rounds. However, our survey response rate of 68.2% is higher than average email response rates amongst physicians, increasing the likelihood that our survey responses captured feedback from most of our critical care physicians who had participated in handshake stewardship rounds . In addiHandshake stewardship is valued as a mode of prospective audit and feedback amongst critical care physicians. Prospective audit and feedback strategies emphasizing face-to-face interaction and bi-directional, verbal feedback may be of greater value compared to other forms of delivering prospective audit and feedback in high-stakes environments such as critical care.


ColIV) and applied them for ultrasound molecular imaging of endothelial injury at early stage. Our results showed that the MBColIV, with a typical multi-peak particle distribution, was successfully constructed, which was confirmed by Alexa Fluor® 555-labeled secondary antibody. Ex vivo adhesion of microbubbles revealed that MBColIV can effectively and specially bind to the surface of balloon-injured carotid artery. The in vivo animal experiments showed ultrasound molecular imaging signals from carotid artery-injured rats administrated with MBColIV were significantly higher than those administrated with isotype control microbubbles. Histological staining of the left carotid common artery revealed that collagen IV was obviously exposed after endothelium denudation in balloon-injured artery. In conclusion, our current study provides an effective approach to detect vascular injury at the early stage and a potential platform for image-guided therapy to vascular injury.Cardiovascular intervention is a common treatment procedure for many cardiovascular diseases. But restenosis often occurs after these procedures, greatly discounting their long-term therapeutic effects. Early detection of endothelial denudation is helpful for the diagnosis and prevention of restenosis. Here, we fabricated targeted microbubbles by conjugating anti-collagen IV antibodies to the surface of biotinylated microbubbles (MB Cardiovascular diseases from atherosclerosis are responsible for the major cause of mortality worldwide , could improve long-term survival and reduce late myocardial infarction and recurrent unstable angina requiring rehospitalization for patients with non-ST-elevation myocardial infarction  and applied them for ultrasound molecular imaging of endothelial injury at early stage . Biotinylated MBs (MBbiotin) were obtained by mechanically vibrating for 30 s. The resulting MBbiotin were washed with phosphate buffered saline (PBS) solution twice to eliminate excess unincorporated lipids by centrifuge at 400 g for 3 min. Three microgram of avidin per 107 MBs was then incubated with the MBbiotin dispersion for 30 min at room temperature, followed by rinsing three times to remove unreacted avidin . Then either 2.5 μg of biotinylated goat anti-rat Type IV Collagen antibody or biotinylated goat IgG isotype control antibody  was added to per 108 MBs. After incubation for 30 min at room temperature, MBColIV or isotype control MBs (MBCtrl) were collected by centrifugation. Non-targeted MBs (MBN) without conjugated primary antibody were also prepared.Biotinylated, lipid-shelled MBs were prepared as previously described .To confirm the conjugation of Type IV Collagen antibody to the surface of MBs, Alexa Fluor® 555-labeled anti-goat secondary antibody  was added to MBColIV, MBN were used as the blank control. The morphology of the MBs were examined by using bright-field and fluorescence inversion microscopy . Particle size, size distribution and concentration of MBs were determined by using the Accusizer 780 Optical Particle Sizer  with a 0.5 μm diameter detection limit.To analyze the characteristics of the MBAll animal procedures were performed in accordance with the principles outlined in the Guide for the Care and Use of Laboratory Animals and approved by Animal Care and Use Committee. Adult male Sprague–Dawley rats , weighing 400–500 g, underwent the rat carotid artery balloon injury as previously described  and transferred to liquid nitrogen for flash-freeze. Vessels were sectioned to obtain arterial cross-sections across the length of the artery by cryostat microtome  and were examined histologically using routine hematoxylin-eosin (H&E) staining for morphometric analysis. Digital images were collected with bright-field by inversion microscopy .Bilateral carotid arteries were harvested following in situ perfusion fixation were completed, the LCCA was harvested and then opened longitudinally. The side of adventitia was adhered to a coverslip. Then MBs were added into a 6-well plate (2 × 107 MBs per well) and filled with PBS. Due to the static flotation nature of MBs, the coverslip with the artery coated faced downward to maximize interaction between the tissue and MBs for 5 min at room temperature. After that, the free MBs were removed by rinsing five times with PBS. To further determine the specificity of MBs adhesion, vessel tissues were pretreated with 25 μg/mL goat anti-rat Type IV Collagen antibody to block available receptors prior to MBColIV incubation. Digital images were obtained with bright-field by inversion microscopy  to count the number of attached MBs in five random fields of view.Attachment ability of MBs to collagen IV was evaluated by counting the number of adhering MBs. Briefly, after the rat carotid artery injury model and the 8 MBs/ml. Then 200 μl MBs suspension was injected intravenously through tail vein following by flushing with 50 μl PBS. Four minutes after MBs injection, 100 frames of images were captured to obtain a signal from adherent and freely circulating MBs. A continuous high-power destructive pulse  was then applied for 2 s to destroy these MBs. After 2 s, to allow the freely circulating MBs to replenish, another 100 frames of images were acquired, in which the ultrasonic signals were from any residual freely circulating MBs and tissue. To minimize the bias and test the specificity of these molecular imaging signals only resulting from adherent targeted MBs, MBColIV, and MBCtrl were administered in random order to all rats. Another 30-min delay was allowed to clear MBs from the preceding imaging session. As a control, ultrasound molecular imaging was also performed in normal rats to further assess the specificity of MBs adhesion. The acoustic imaging signals were analyzed by using commercially available analysis software . As previously described  using a L11-3 linear array transducer. Contrast imaging mode was applied in the ultrasound molecular imaging experiments. All imaging parameters were kept constant throughout the whole procedure as follows: frequency 5.6 MHz, depth 2 cm, gain 45 dB, frame rate 10 Hz, dynamic range 115 dB, and mechanical index 0.085. In order to reduce motion interference, both the transducer and the rats were fixed to maintain the same long axis cross section of carotid artery. The concentration of MBs suspension was adjusted to 2 × 10in situ perfusion fixation with PBS and 4% paraformaldehyde. Then artery samples were transferred to paraformaldehyde overnight, followed by ethanol dehydration and paraffin embedment. Artery samples were sectioned to obtain arterial cross-sections. After the process of deparaffinage and rehydration, slides were blocked and then incubated with the primary antibody goat anti-rat Type IV Collagen , followed by being rinsed with PBS thrice and then incubated with horseradish peroxidase-conjugated secondary antibodies. Finally, slides were incubated with diaminobenzidine for horseradish peroxidase reaction. In order to exhibit the tissue morphology of vascular wall, slides were counterstained with hematoxylin. Immunohistochemical images were acquired by using inversion microscopy  under bright field.Immunostaining analysis for collagen IV was performed to confirm that collagen IV was exposed through endothelium denudation. Briefly, LCCAs from normal artery group and balloon-injured artery group were excised, followed by t-test, and one-way ANOVA were used to compare among more than two groups. If the differences among these groups were significant for one-way ANOVA, the difference between the two groups was conducted by Student-Newman-Keuls test. The P < 0.05 indicated statistically significant difference.Data were analyzed by the statistic software SPSS 20.0 . As values were normally distributed, statistic difference between two groups was determined by Student's ColIV appeared a typical multi-peak particle distribution. There was no significant difference between MBColIV and MBN for the particle size and distribution . The mean diameter of MBColIV and MBN were 1.92 and 1.89 μm, respectively. The results demonstrated that the conjunction of avidin and biotinylated antibody to MBs surface did not change the particle size and distribution of MBs   Figure .in vivo, ultrasound molecular imaging was performed in the balloon-injured carotid artery group (n = 8) and the normal carotid artery group (n = 7). As shown in Figure ColIV were hardly attached to the normal artery wall  was higher than MBCtrl (0.57 ± 0.26 dB) in balloon-injured carotid artery group (P < 0.01)   Figure . Moreove) Figure .ColIV. In addition, collagen IV was also a component of the basement membrane for SMCs in tunica media, which had been reported in previous studies (Shekhonin et al., To confirm the results of ultrasound molecular imaging, LCCAs of the normal group and balloon-injured group were harvested and analyzed for the exposure of collagen IV after endothelium denudation. The results of immunohistochemical staining examination were presented in Figure Microbubble-based ultrasound molecular imaging is a promising approach in the disease diagnosis at the early stage. This is especially the case for vascular disorders since the microscale-targeted bubbles only exist in the blood and are not extravasate from the circulation. This feature makes it greatly standing out from other nanoparticle-based molecular imaging modalities, such as quantum dot-based optical molecular imaging and iron nanoparticle-based magnetic imaging resonance (Lobatto et al., ColIV by biotin-avidin bridging method. As shown in Figure in vitro tool in our static binding experiments such as culture plates, pre-coated with Matrigel, which has been taken to examine the binding of targeted particles to collagen IV (Dong et al., ex vivo binding experiments (Figure ColIV binding to the tissue of injured artery was nearly four times higher than MBCtrl, indicating MBColIV we obtained can be used as a potential ultrasound molecular imaging probe.In our study, we fabricated the targeted MBs Figure . The numin vivo in order to determine whether MBColIV could adhere to the site of endothelial denudation and be visually detected by a clinical ultrasound scanner or not. Surprisingly, we found stronger ultrasound molecular imaging signals from injured vascular wall after MBColIV injection but not MBCtrl, the former being nearly five times higher than MBCtrl. Interestingly, no obvious ultrasound molecular imaging signals were detected from the normal arterial wall after MBColIV or MBCtrl injection. These results suggested that MBColIV had a highly specific binding ability to these collagen IV targets and could function as an ultrasound molecular imaging probe to detect vascular injury. To take the drug-loaded advantages of MBs into consideration, it is easy to design the MBColIV into drug-loaded targeted MBs through carrying drugs (Lammertink et al., ColIV could be further functionalized as theranostics agent and would play a great role for ultrasound imaging-guided treatment of vascular injury.In our present study, we used intravenously injection for ultrasound molecular imaging There are several limitations in our study. The rat carotid artery balloon injury model described in our study imitate vascular injury by angioplasty. However, in clinical practice, restenosis occurs in patients who have occlusive atherosclerotic lesions after dilating the narrow lumen through angioplasty. Besides, to evaluate non-culprit stenosis severity, the instantaneous wave-free ratio, which is helpful for guiding PCI strategy, has been regarded as the promising method and used in patients with acute coronary syndrome and multivessel disease (Indolfi et al., ColIV by conjugating anti-collagen IV antibodies to the surface of MBs and confirmed the feasibility to apply them for ultrasound molecular imaging of endothelial injury at early stage. Our study provides an early diagnosis tool for restenosis due to endothelial damage and a potential platform for image-guided therapy for vascular injury.In summary, we have successfully fabricated MBXM and BZ initialed the project. XM and FY conducted the experiment and data analysis. BZ and FY provided equipment, materials, and experimental methods. XM wrote the manuscript. FY proofread the manuscript. All authors agreed with the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Transtibial amputees may experience decreased quality of life due to increased risk of knee joint osteoarthritis (OA). No prior studies have compared knee joint biomechanics for the same group of transtibial amputees in gait, cycling, and elliptical training. Thus, the goal of this study was to identify preferred exercises for transtibial amputees in the context of reducing risk of knee OA. The hypotheses were: 1) knee biomechanics would differ due to participant status , exercise, and leg type  and 2) gait kinematic parameters would differ due to participant status and leg type. Ten unilateral transtibial amputee and ten control participants performed exercises while kinematic and kinetic data were collected. Two-factor repeated measures analysis of variance with post-hoc Tukey tests and non-parametric equivalents were performed to determine significance. Maximum knee compressive force, extension torque, and abduction torque were lowest in cycling and highest in gait regardless of participant type. Amputee maximum knee extension torque was higher in the intact vs. residual knee in gait. Amputee maximum knee flexion angle was higher in the residual vs. intact knee in gait and elliptical. Gait midstance knee flexion angle timing was asymmetrical for amputees and knee angle was lower in the amputee residual vs. control non-dominant knees. The results suggest that cycling, and likely other non-weight bearing exercises, may be preferred exercises for amputees due to significant reductions in biomechanical asymmetries and joint loads. Transtibial amputees may experience decreased quality of life due to increased risk of knee joint osteoarthritis (OA) , a degenin vivo study showed that extension torque is a significant contributor to medial contact force [Transtibial amputees are more likely to develop OA in the intact knee joint than the residual (i.e. amputated) joint ,11,12, wct force .While prosthetic limb design has advanced in recent years, with energy storage and release (ESAR) prostheses representing the state-of-the-art in passive-elastic devices, such prostheses do not completely restore natural biomechanics of the lower limb during gait –30. StudExercise is recommended for amputees for rehabilitation and lifelong fitness sustainment  but therThe goal of this study was to conduct motion analysis experiments and compare knee biomechanics in gait, cycling, and elliptical training in order to identify appropriate exercises for transtibial amputees whom are at high risk for knee OA. The hypotheses were: 1) knee biomechanics would differ due to participant status , exercise, and leg type  and 2) gait kinematic parameters would differ due to participant status and leg type.All protocols were approved by Cal Poly's Institutional Review Board (IRB) and the U.S. Army Medical Research and Materiel Command (USAMRMC) Office of Research Protections (ORP) Human Research Protections Office (HRPO) (HRPO Log Number A-19263) and were designed to minimize risk to human subjects. Written consent was obtained prior to all experiments.®, Ӧssur, Reykjavik, Iceland) by a certified prosthetist (AMR). One week of accommodation time was provided to amputee participants that exhibited unfamiliarity with the Vari-Flex® as determined by our certified prosthetist (AMR) in qualitative alignment and gait pattern checks. However, most (8/10) amputee participants were tested without accommodation time as they used similar ESAR devices to the Vari-Flex® and passed AMR’s alignment and gait pattern checks. Amputee participants all wore similar carbon fiber sockets and static pylons with no springs or dampers. Screening and health information forms indicated that all participants were relatively young, healthy, non-obese, free of disease, and had no restrictions on physical activity.Ten unilateral transtibial amputees  and ten control participants  participated. Exclusion criteria included history of cardiovascular disease, respiratory disease, or any other metabolic disease/complication; substantial weight loss or gain over the previous six months; history of major psychiatric illness, drug abuse, or unsafe dieting practices; major medical conditions that prohibit physical activity; and pregnant women or women expecting or trying to be pregnant. After screening, eligible participants were invited to the motion analysis lab where informed consent was obtained, information forms were filled out, and amputee participants were fitted with an Energy Storage and Release (ESAR) prosthesis   were used capture the motion of reflective markers. Participants wore tight compression clothing that exposed as much skin as the participant was comfortable with. A modified Helen Hayes marker set was used with 32 reflective markers placed at the following anatomical landmarks: crown of the head, acromion processes, 7Participants were asked to stand stationary in a static pose with feet at shoulder width and arms bent at the elbows. Static pose captures were used to begin the marker identification process and defined initial joint angles for later analyses. Top head and medial knee and ankle markers were removed after static captures. During gait experiments, four ground force plates  were used to capture ground reaction forces in the anterior, lateral, and vertical directions; the free reaction torque about the vertical axis; and the center of pressure (COP) of the reaction force vector on the surface of the force platform . Three gDuring cycling and elliptical experiments, the pedals of a stationary bicycle  and elliptical trainer  were instrumented with 6-axis load cells  to capture pedal reaction forces, free torque, and COP . Three tst heel strike, 100% = 2nd heel strike). One cycling cycle was defined by one full crank rotation starting at top dead center  [st pass through APP, 100% = 2nd pass through APP) [One full gait cycle was defined by consecutive heel strikes of the same leg (0% = 1ugh TDC) . One ellugh APP) .The sacrum marker velocity in the direction of motion was stored and used to calculate average walking speed for each participant. The time in seconds at which cycling and elliptical pedals passed through their respective start and end of cycle were stored and used to calculate actual RPM. Cycles with RPM greater than 72 or less than 68 were eliminated from the pool of samples as kinetics vary with machine power [Static and dynamic captures were processed in Cortex to define body segments used to calculate joint kinematics and kinetics. For cycling and elliptical trials, additional markers defined pedal segments which were necessary to track load cells relative to a global coordinate system and transform measured forces and torques to the foot coordinate system . For theth order, zero phase shift Butterworth filter with a cutoff frequency of 6 Hz and reaction forces were filtered using the same filter with a 10 Hz cutoff frequency. Cutoff frequency for reaction forces was calculated using an optimal method [Kinematic and kinetic data were captured in Cortex at 150 Hz. Marker trajectories were filtered in Cortex using a two-pass, 4l method . MissingKinetics were calculated in Cortex using a bottom-up approach. The Cortex package KinTools RT used lower-body kinematics, standard shank and foot segment inertial properties , and meaAnalyses of kinematic and kinetic data results were performed using a custom script in MATLAB . All dynamic trials were interpolated to 101 data points from 0% to 100% cycle. Three trials for each participant in each exercise for each of the intact/dominant and residual/non-dominant legs were averaged. Knee kinematics and kinetics were then averaged across ten participants in each participant group.Maximum knee flexion angles in gait, cycling, and elliptical for the intact/dominant and residual/non-dominant legs were normalized for each participant group by subtracting calculated knee joint flexion angles of the static pose from the dynamic angles measured during each trial . Also, mAnalyzed data were tested for normality using Shapiro-Wilks tests and for equal variance using F-tests. Maximum knee compression force, extension torque, abduction torque, and maximum swing knee flexion angle in gait were found to be non-normal distributions and were analyzed using Kruskal-Wallis tests and post-hoc Dunn tests at 95% confidence with Bonferroni adjustments for multiple comparisons to analyze the differences between participant-leg-exercise groupings. Two-factor repeated measures analysis of variance (ANOVA) with post-hoc Tukey tests at 95% confidence were conducted to analyze the effects of participant and leg type  and exercise  on maximum knee flexion angle and to analyze the effects of participant and leg type  on knee flexion angle peaks and peak times in gait. All statistical analyses were performed in R . A BonfeThe average amputee and control participant walking speeds were 1.26 ± 0.17 m/s and 1.29 ± 0.08 m/s, respectively. Average cycling RPMs were 69.45 ± 0.61 and 69.75 ± 0.61 for amputees and controls, respectively. Average elliptical RPMs were 70.32 ± 0.59 and 70.15 ± 0.54 for amputees and controls, respectively. A two-sample t-test showed that the walking speeds were not different (p = 0.566) between participant groups (t-tests were only run on gait speeds since they were self-selected).Maximum knee flexion angles were different between exercise types for all participant groups . P-valueMaximum knee compressive forces varied in gait vs. cycling for all participant types and in cycling vs. elliptical for Amp-Intact legs . P-valueMaximum knee internal extension torque was different in gait vs. cycling for Amp-Intact and Con-ND legs and in cycling vs. elliptical for Amp-Intact legs . P-valueMaximum knee internal abduction torque was different in gait vs. cycling for all participant types . P-valueP-value for maximum midstance knee flexion angle in gait was 0.005 for Amp-Residual vs. Con-ND legs . P-valueThe hypotheses of the current study were: 1) knee biomechanics would differ due to participant status , exercise, and leg type  and 2) gait kinematic parameters would differ due to participant status and leg type. Kinematics and kinetics varied with exercise , leg type , and participant type (extension torque in gait) providing support for the first hypothesis. Gait characteristics varied with leg type  and participant type  providing support for the second hypothesis. Knee joint compressive force, torques, and flexion angles were of similar magnitude to many other studies in both amputee and non-amputee populations in gait ,17,21,54The kinematic results of the present study have clinical implications regarding rehabilitative and lifelong fitness sustainment exercises that may minimize OA risk in amputees. In the general population, abnormal knee kinematics are linked to the initiation of knee OA ,58. SpecAlso, the kinetic results of the present study have similar clinical implications. Knee kinetics such as compressive force and muscle torques can be an indication of OA severity ,61; specThe results of the current study suggest that cycling may be an appropriate exercise for transtibial amputees whom are at high risk for knee OA. Specifically, the results suggest that exercises that constrain kinematics, such as cycling, are more likely to maintain typical cartilage loading patterns due to a lack of knee flexion angle and extension torque asymmetry as compared to gait and elliptical. The results also suggest that exercises that reduce overall knee joint forces and torques may be preferred for reducing OA risk. Cycling had generally lower magnitudes of resultant knee compressive force, extension torque, and abduction torque compared to elliptical training and gait. Other non-weight bearing exercises such as rowing or non-impact exercises such as stair-stepping may also be preferred over gait, while high-impact activities such as running and sports that involve foot planting (such as soccer) may increase the rate of OA progression due to high knee joint compression forces. Exercises for rehabilitation should, in general, minimize kinematic abnormalities and joint compressive stress to prevent or alleviate abnormal loading of cartilage but the present study did not relate exercise type to injury or OA risk. There is a clear need for a long-term study to associate injury risk with varying exercise protocols to produce specific exercise guidelines for limiting OA risk.There are several limitations to the current study. One limitation of this work included marker-based errors which are common in motion analysis. Markers may have moved relative to the underlying bones due to soft tissue artifact (STA) and relative motion of compression clothing. STA, caused by marker movement due to skin deformation and displacement, affects the estimation of skeletal system kinematics with the exception of motion about joint flexion-extension axes ,67; thisA second limitation was inherent issues in any prosthetic assembly for amputee participants. While the same ESAR foot was used for all amputee participants the socket was not modified to enhance user experience and comfort with the prosthesis in exercises that amputee participants were not familiar with . Socket-limb interfaces  can permit motion of the prosthesis relative to the residual limb which can affect joint biomechanics. Future studies should make efforts to measure prosthesis motion relative to the residual limb.In conclusion, transtibial amputees had significant asymmetry between intact and residual knee flexion angle in gait and elliptical and significantly reduced extension torque in the residual vs. intact knee in gait, whereas no asymmetries were detected for transtibial amputees in cycling. The results suggest that cycling, and likely other non-weight bearing exercises, may be a preferred exercise for limiting OA risk in transtibial amputees due to reduced asymmetry in knee kinematics and reduced knee kinetics as compared to gait values. Also, the results showed that state-of-the-art ESAR prosthetic design may not sufficiently restore amputee biomechanics to normal levels as evidenced by midstance knee flexion angle and peak extension torque asymmetry in gait and peak knee flexion angle asymmetry in elliptical. Muscle activation patterns could be responsible for altered residual knee flexion angle and extension torque in amputees and future work should involve EMG-driven inverse dynamics to calculate intact knee joint contact forces and muscle contributions. Since this study did not directly relate exercise type to injury or OA risk, there is a need for a long-term study that aims to directly associate injury or OA risk with varying exercise protocols for transtibial amputees and other populations at high risk for knee OA.


Dynapenia or muscle weakness is a core feature of sarcopenia and frailty, and leads to significant functional impairment in older adults. We aimed to identify genetic variants associated with dynapenia in older adults, to shed light on the underlying mechanisms. A large-scale meta-analysis of >250,000 volunteers of European descent from 16 cohorts in the CHARGE and GEFOS consortia aged 60+ years at assessment is underway, using maximum hand grip strength to define dynapenia. Preliminary analysis in UK Biobank found that dynapenia is associated with nine genomic loci at genome wide significance (p<5x10-8), with differences between men and women, suggesting sex-specific routes to dynapenia. These results suggest that there are specific genetic determinants of low strength in older adults, and these differ from the determinants of normal-range strength differences. The full meta-analysis of all cohorts will extend these findings. Understanding the biological mechanisms may lead to geroscience interventions.