Establishment and Validation of Pre-Therapy Cervical Vertebrae Muscle Quantification as a Prognostic Marker of Sarcopenia in Patients With Head and Neck Cancer.

BACKGROUND: Sarcopenia is prognostic for survival in patients with head and neck cancer (HNC). However, identification of this high-risk feature remains challenging without computed tomography (CT) imaging of the abdomen or thorax. Herein, we establish sarcopenia thresholds at the C3 level and determine if C3 sarcopenia is associated with survival in patients with HNC. METHODS: This retrospective cohort study was conducted in consecutive patients with a squamous cell carcinoma of the head and neck with cross-sectional abdominal or neck imaging within 60 days prior to treatment. Measurement of cross-sectional muscle area at L3 and C3 levels was performed from CT imaging. Primary study outcome was overall survival. RESULTS: Skeletal muscle area at C3 was strongly correlated with the L3 level in both men (n = 188; r = 0.77; p < 0.001) and women (n = 65; r = 0.80; p < 0.001), and C3 sarcopenia thresholds of 14.0 cm2/m2 (men) and 11.1 cm2/m2 (women) were best predictive of L3 sarcopenia thresholds. Applying these C3 thresholds to a cohort of patients with neck imaging alone revealed that C3 sarcopenia was independently associated with reduced overall survival in men (HR = 2.63; 95% CI, 1.79, 3.85) but not women (HR = 1.18, 95% CI, 0.76, 1.85). CONCLUSIONS: This study identifies sarcopenia thresholds at the C3 level that best predict L3 sarcopenia in men and women. In HNC, C3-defined sarcopenia is associated with poor survival outcomes in men, but not women, suggesting sarcopenia may differentially affect men and women with HNC.

Rapid Detection and Purification of Galectin-3 by the Capture and Release (CaRe) Method.

Specific interactions between lectins and glycoproteins determine the outcomes of numerous biological processes. To elucidate the roles of lectins and glycoproteins in those processes, it is essential to detect these proteins in biological samples and purify them to homogeneity. Conventional protein detection and purification techniques are multi-step, time-intensive, and expensive. They often require rigorous trial and error experimentations and fairly larger volumes of crude extracts. To minimize some of these challenges, we recently formulated a new method named Capture and Release (CaRe). This method is rapid, facile, precise, and inexpensive, and it works even when the sample volume is smaller. We developed this method to detect and purify recombinant human Galectin-3 and subsequently validated this method by purifying several other lectins. Besides lectins, CaRe is capable of detecting/purifying glycoproteins. In this method, targets (lectins and glycoproteins) are captured by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. CaRe can potentially be used as a tool to discover new lectins and glycoconjugates and elucidate their functions.

Revealing the Identity of Human Galectin-3 as a Glycosaminoglycan-Binding Protein.

Human galectin-3 (Gal-3) is a ß-galactoside-binding lectin. This multitasking protein preferentially interacts with N-acetyllactosamine moieties on glycoconjugates. Specific hydroxyl groups (4-OH, 6-OH of galactose, and 3-OH of glucose/N-acetylglucosamine) of lactose/LacNAc are essential for their binding to Gal-3. Through hemagglutination inhibition, microcalorimetry, and spectroscopy, we have shown that despite being a lectin, Gal-3 possesses the characteristics of a glycosaminoglycan (GAG)-binding protein (GAGBP). Gal-3 interacts with sulfated GAGs [heparin, chondroitin sulfate-A (CSA), -B (CSB), and -C (CSC)] and chondroitin sulfate proteoglycans (CSPGs). Heparin, CSA, and CSC showed micromolar affinity for Gal-3, while the affinity of CSPGs for Gal-3 was much higher (nanomolar). Interestingly, CSA, CSC, and a bovine CSPG, not heparin and CSB, were multivalent ligands for Gal-3, and they formed reversible noncovalent cross-linked complexes with the lectin. Binding of sulfated GAGs to Gal-3 was completely inhibited when Gal-3 was preincubated with ß-lactose. Cross-linking of Gal-3 by CSA, CSC, and the bovine CSPG was also reversed by ß-lactose. These findings strongly suggest that GAGs primarily occupy the lactose/LacNAc binding site of Gal-3. Identification of Gal-3 as a GAGBP should help to reveal new functions of Gal-3 mediated by GAGs and proteoglycans. The GAG- and CSPG-binding properties of Gal-3 make the lectin a potential competitor/collaborator of other GAGBPs such as growth factors, cytokines, morphogens, and extracellular matrix proteins.

Mechanism of Mucin Recognition by Lectins: A Thermodynamic Study.

Isothermal titration microcalorimetry (ITC) can directly determine the thermodynamic binding parameters of biological molecules including affinity constant, binding stoichiometry, heat of binding (enthalpy) and indirectly the entropy, and free energy of binding. ITC has been extensively used to study the binding of lectins to mono- and oligosaccharides, but limitedly in applications to lectin-glycoprotein interactions. Inherent experimental challenges to ITC include sample precipitation during the experiment and relative high amount of sample required, but careful design of experiments can minimize these problems and allow valuable information to be obtained. For example, the thermodynamics of binding of lectins to multivalent globular and linear glycoproteins (mucins) have been described. The results are consistent with a dynamic binding mechanism in which lectins bind and jump from carbohydrate to carbohydrate epitope in these molecules leading to increased affinity. Importantly, the mechanism of binding of lectins to mucins appears similar to that for a variety of protein ligands binding to DNA. Recent results also show that high-affinity lectin-mucin cross-linking interactions are driven by favorable entropy of binding that is associated with the bind and jump mechanism. The results suggest that the binding of ligands to biopolymers, in general, may involve a common mechanism that involves enhanced entropic effects that facilitate binding interactions.

Effects of prolonged peri-neural bupivacaine infusion in rat sciatic nerves (axon and myelin).

BACKGROUND/OBJECTIVE: Peripheral-nerve blocks (PNBs) using continuous-infusion of local anesthetics are used to provide perioperative analgesia. Yet little research exists to characterize the histopathological effects of continuous long-duration PNBs. Herein we test the hypothesis that continuous peri-neural bupivacaine infusion (3-day vs. 7-day infusion) contributes to histologic injury in a duration-dependent manner using an in vivo model of rat sciatic nerves. METHODS: We placed indwelling catheters in 22 rats for infusion with low-dose (0.5mg/kg/hr) bupivacaine or normal saline proximal to the right sciatic nerves for 3 or 7 consecutive days. Hind-limb analgesia was measured using Von-Frey nociceptive testing. At infusion end, rats were sacrificed, bilateral nerves were sectioned and stained with hematoxylin and eosin and CD68 for evaluation of inflammatory response, and eriochrome to assess damage to myelin. RESULTS: Animals receiving continuous infusion of bupivacaine maintained analgesia as demonstrated by significant decrease (50% on average) in nociceptive response in bupivacaine-infused limbs across time points. Both 7-day saline and bupivacaine-infused sciatic nerves showed significantly-increased inflammation by H&E staining compared to untreated native nerve controls (P = 0.0001, P < 0.0001). Extent of inflammation did not vary significantly based on infusate (7-day saline vs. 7-day bupivacaine P > 0.99) or duration (3-day bupivacaine vs 7-day bupivacaine P > 0.99). No significant change in sciatic nerve myelin was found in bupivacaine-infused animals compared to saline-infused controls, regardless of duration. CONCLUSIONS: Long-duration (7-day) bupivacaine infusion provided durable post-operative analgesia, yet contributed to equivalent neural inflammation as short duration (3-day) infusion of bupivacaine or saline with no evidence of demyelination.

Lipocalin 2 mediates appetite suppression during pancreatic cancer cachexia.

Lipocalin 2 (LCN2) was recently identified as an endogenous ligand of the type 4 melanocortin receptor (MC4R), a critical regulator of appetite. However, it remains unknown if this molecule influences appetite during cancer cachexia, a devastating clinical entity characterized by decreased nutrition and progressive wasting. We demonstrate that LCN2 is robustly upregulated in murine models of pancreatic cancer, its expression is associated with reduced food consumption, and Lcn2 deletion is protective from cachexia-anorexia. Consistent with LCN2's proposed MC4R-dependent role in cancer-induced anorexia, pharmacologic MC4R antagonism mitigates cachexia-anorexia, while restoration of Lcn2 expression in the bone marrow is sufficient in restoring the anorexia feature of cachexia. Finally, we observe that LCN2 levels correlate with fat and lean mass wasting and is associated with increased mortality in patients with pancreatic cancer. Taken together, these findings implicate LCN2 as a pathologic mediator of appetite suppression during pancreatic cancer cachexia.

Bioelectrical impedance analysis as a quantitative measure of sarcopenia in head and neck cancer patients treated with radiotherapy.

BACKGROUND AND PURPOSE: Sarcopenia is associated with decreased survival in head and neck cancer patients treated with radiotherapy. This study sought to determine whether in-clinic multifrequency bioelectrical impedance analysis (BIA) can identify survival-associated sarcopenia in patients with head and neck cancer. MATERIALS AND METHODS: This prospective observational study enrolled 50 patients with head and neck cancer undergoing radiation therapy. Baseline BIA measures of skeletal muscle (SM) mass, fat-free mass (FFM), and fat mass (FM) were compared to CT-based estimates using linear regression. Sex-specific BIA-derived thresholds for sarcopenia were defined by the maximum Youden Index on receiver operator characteristic (ROC) curves. Patients were stratified by sarcopenia status and OS was compared using the Kaplan-Meier method and log-rank test. RESULTS: Among 48 evaluable patients, BIA measures of body composition were strongly correlated with CT measures: SM mass (r = 0.97; R2 = 0.94; p < 0.0001), FFM (r = 0.97; R2 = 0.94; p < 0.0001) and FM (r = 0.95; R2 = 0.90; p < 0.0001). SM mass index < 9.19 kg/m2 identified sarcopenia men with high sensitivity (91.7%) and specificity (92.9%), whereas in women SM mass index < 6.53 kg/m2 was sensitive for sarcopenia (100%), but not specific. Patients with sarcopenia, defined by either CT or BIA, exhibited decreased OS (HR = not estimable; CT p = 0.009; BIA p = 0.03). CONCLUSION: BIA provides accurate estimates of body composition in head and neck cancer patients. Implementation of BIA in clinical practice may identify patients with sarcopenia at risk for poor survival.

A Rapid and Facile Purification Method for Glycan-Binding Proteins and Glycoproteins.

Glycosylated proteins, namely glycoproteins and proteoglycans (collectively called glycoconjugates), are indispensable in a variety of biological processes. The functions of many glycoconjugates are regulated by their interactions with another group of proteins known as lectins. In order to understand the biological functions of lectins and their glycosylated binding partners, one must obtain these proteins in pure form. The conventional protein purification methods often require long times, elaborate infrastructure, costly reagents, and large sample volumes. To minimize some of these problems, we recently developed and validated a new method termed capture and release (CaRe). This method is time-saving, precise, inexpensive, and it needs a relatively small sample volume. In this approach, targets (lectins and glycoproteins) are captured in solution by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. Application of the CaRe method could play an important role in discovering new lectins and glycoconjugates. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of crude extracts containing the target proteins from soybean flour Alternate Protocol 1: Preparation of crude extracts from Jack bean meal Alternate Protocol 2: Preparation of crude extracts from the corms of Colocasia esculenta, Xanthosoma sagittifolium, and from the bulbs of Allium sativum Alternate Protocol 3: Preparation of Escherichia coli cell lysates containing human galectin-3 Alternate Protocol 4: Preparation of crude extracts from chicken egg whites (source of ovalbumin) Basic Protocol 2: Preparation of 2% (v/v) red blood cell suspension Basic Protocol 3: Detection of lectin activity of the crude extracts Basic Protocol 4: Identification of multivalent inhibitors as target capturing agents by hemagglutination inhibition assays Basic Protocol 5: Testing the capturing abilities of target capturing agents by precipitation/turbidity assays Basic Protocol 6: Capturing of targets (lectins and glycoproteins) in the crude extracts by target capturing agents and separation of the target-TCA complex from other components of the crude extracts Basic Protocol 7: Releasing the captured targets (lectins and glycoproteins) by dissolving the complex Basic Protocol 8: Separation of the targets (lectins and glycoproteins) from their respective target capturing agents Basic Protocol 9: Verification of the purity of the isolated targets (lectins or glycoproteins).

Association of Sarcopenia With Oncologic Outcomes of Primary Surgery or Definitive Radiotherapy Among Patients With Localized Oropharyngeal Squamous Cell Carcinoma.

Importance: The negative association of low lean muscle mass (sarcopenia) with survival outcomes in head and neck cancers, including oropharyngeal carcinoma, is established. However, it is not known whether the choice of primary treatment modality (surgery or radiotherapy) is associated with oncologic outcomes of patients with sarcopenia and oropharyngeal squamous cell carcinoma (OPSCC). Objective: To examine whether primary surgical resection or definitive radiotherapy is associated with improved survival for patients with sarcopenia and localized OPSCC. Design, Setting, and Participants: A cohort study was conducted of patients with clinically staged T1 to T2, N0 to N2 OPSCC with cross-sectional abdominal imaging within 60 days prior to treatment and treated between January 1, 2005, and December 31, 2017. Skeletal muscle mass was measured at the third lumbar vertebra using previously defined techniques and sarcopenia was defined as less than 52.4 cm2/m2 of muscle for men and less than 38.5 cm2/m2 for women. In addition, associated patient demographic characteristics, cancer data, treatment information, and survival outcomes were assessed. Statistical analysis was performed from December 3, 2018, to August 28, 2019. Main Outcomes and Measures: Primary outcomes were overall survival and disease-specific survival. Results: Among the 245 patients who met study inclusion criteria, 209 were men (85.3%) and the mean (SD) age was 62.3 (7.8) years. Sarcopenia was detected in 135 patients (55.1%), while normal skeletal muscle mass was detected in 110 patients (44.9%). For the 110 patients without sarcopenia, primary treatment modality was not associated with improved survival. For patients with sarcopenia at diagnosis, primary surgical resection was associated with improved overall survival (hazard ratio [HR], 0.37; 95% CI, 0.17-0.82) and disease-specific survival (HR, 0.22; 95% CI, 0.07-0.68). This association persisted after propensity score matching, as up-front surgery was associated with improved overall survival (HR, 0.33; 95% CI, 0.12-0.91) and disease-specific survival (HR, 0.17; 95% CI, 0.04-0.75) survival. Conclusions and Relevance: This study suggests that sarcopenia has a negative association with survival for patients with OPSCC. Primary surgery and radiotherapy confer similar survival associations for patients with normal skeletal muscle mass and localized OPSCC. However, up-front surgical resection may be associated with improved survival outcomes for patients with sarcopenia.

Co-presentation of pulmonary arteriovenous malformation (PAVM) and multifocal benign metastasizing leiomyoma (BML) of lungs: Diagnostic dilemma and successful endovascular treatment.

A 50-year-old female with a 20-year history of multifocal pulmonary benign metastasizing leiomyoma (BML), and asthma presented with subacute worsening of chronic dyspnea. A contrast-enhanced computerized tomography of the chest showed a single 1.4 × 1.5-cm contrast-enhancing mass in the right lower lobe among numerous non-enhancing bilateral pulmonary BML lesions. Pulmonary angiogram was not performed at that time due to clinical improvement. Four years later, the patient presented with refractory subacute worsening of her chronic dyspnea and was referred for embolization of the pulmonary arteriovenous malformation (PAVM). Two feeder arteries to the PAVM were embolized; each with a 6-mm Amplatzer-IV vascular plug and a 4-mm Nester coil. Follow-up angiograms demonstrated no flow through the PAVM. The patient's dyspnea resolved and she remained asymptomatic at one-year follow-up.

A capture and release method based on noncovalent ligand cross-linking and facile filtration for purification of lectins and glycoproteins.

Glycan-binding proteins such as lectins are ubiquitous proteins that mediate many biological functions. To study their various biological activities and structure-function relationships, researchers must use lectins in their purest form. Conventional purification techniques, especially affinity column chromatography, have been instrumental in isolating numerous lectins and glycoproteins. These approaches, however, are time-consuming, consist of multiple steps, and often require extensive trial-and-error experimentation. Therefore, techniques that are relatively rapid and facile are needed. Here we describe such a technique, called capture and release (CaRe). The strength of this approach is rooted in its simplicity and accuracy. CaRe purifies lectins by utilizing their ability to form spontaneous noncovalently cross-linked complexes with specific multivalent ligands. The lectins are captured in the solution phase by multivalent capturing agents, released by competitive monovalent ligands, and then separated by filtration. CaRe does not require antibodies, solid affinity matrices, specialized detectors, a customized apparatus, controlled environments, or functionalization or covalent modification of reagents. CaRe is a time-saving procedure that can purify lectins even from a few milliliters of crude protein extracts. We validated CaRe by purifying recombinant human galectin-3 and five other known lectins and also tested CaRe's ability to purify glycoproteins. Besides purifying lectins and glycoproteins, CaRe has the potential to purify other glycoconjugates, including proteoglycans. This technique could also be used for nonlectin proteins that bind multivalent ligands. Given the ubiquity of glycosylation in nature, we anticipate that CaRe has broad utility.