The utility of fine needle aspiration to identify unusual pathology in a parapharyngeal mass

The parapharyngeal space is a complex and well-defined anatomical zone lying lateral to the pharynx and medial to the ramus of the mandible. Although tumors of this space are rare, the parapharyngeal space is difficult to examine clinically; and diagnostic modalities of computerized tomographic scanning and magnetic resonance imaging are primarily used in the evaluation of parapharyngeal space lesions. We present a case report of a second branchial cleft sinus of the parapharyngeal space diagnosed with the assistance of fine needle aspiration (FNA), and we recommend FNA of parapharyngeal masses to provide definitive preoperative diagnoses.     

Yield of Neck CT and Barium Esophagram in Patients with Globus Sensation

BACKGROUND AND PURPOSE:Globus sensation is common and difficult to treat. The purpose of our study was to compare the diagnostic and therapeutic efficacy of barium esophagram and neck CT in patients with isolated globus sensation, to determine which of these modalities should be preferred in the evaluation of this condition.MATERIALS AND METHODS:We retrospectively identified patients presenting with isolated globus sensation from January 1, 2005, to December 31, 2012, who underwent neck CT or barium esophagram. We calculated the proportion of patients with abnormal findings, tabulated the nature of the abnormality, and reviewed the medical records to determine whether imaging changed management.RESULTS:One hundred forty-eight neck CTs and 104 barium esophagrams were included. Five (3.4%) patients with neck CTs and 4 (3.9%) with barium esophagrams demonstrated significant findings related to the history of globus sensation. Of these, 1 (0.7%) neck CT and 1 (1.0%) barium esophagram resulted in a change in clinical management.CONCLUSIONS:Imaging evaluation of the patient with uncomplicated globus sensation is unlikely to identify clinically significant imaging findings and is very unlikely to result in a change in clinical management, with a combined therapeutic efficacy of 0.8%. Thus, the routine use of imaging in the evaluation of patients with globus sensation cannot be recommended.

Globus sensation (GS), an intermittent or persistent painless sensation of a foreign body or lump in the throat, is a long-lasting and often frustratingly difficult-to-treat clinical entity.¹ It is a relatively common condition, accounting for up to 4% of new referrals to otolaryngology clinics, with a prevalence of up to 35% in males and over 50% in females, with a relative peak in middle age.^2–4 A range of etiologies has been suggested and described, including lingual and tonsillar hypertrophy, psychogenic factors, cervical osteophytes, upper aerodigestive tract malignancy, thyroid disease, and esophageal motor disorders.^5–8 More recently, there has been increasing focus on gastroesophageal reflux disease as a cause of GS.^9–13 The myriad potential etiologies of GS have made it difficult to establish standard treatment and imaging strategies for affected patients.The imaging approach to the patient with GS varies widely in clinical practice. A neck CT, usually ordered with contrast, is well-suited to detect many structural causes of GS and is a useful tool to exclude a large upper aerodigestive tract malignancy, while a barium esophagram is well-suited for detailed evaluation of esophageal motility and mucosal and submucosal lesions of the esophagus. While a barium esophagram may also detect (but cannot exclude) intermittent esophageal reflux, if evaluation for esophageal reflux is of primary concern, then esophageal manometry, endoscopy, esophageal pH monitoring, or a trial of empiric therapy is the preferred diagnostic test.^14–16The imaging approach to the patient with GS varies widely in clinical practice. Because an evidence-based approach to imaging GS is lacking in current clinical practice, practitioner and locoregional biases strongly influence the decision to use neck CT or barium esophagram. This may adversely impact the clinical value of these studies because the value of a diagnostic test is largely dependent on the prevalence (or the clinician''s estimate of the pretest probability) of the target disorder, and abnormalities detectable on neck CT and barium esophagram are statistically unlikely etiologies in a general sample of patients with GS. Because overuse of diagnostic tests contributes to both the rising cost and the overall quality of health care, defining the value of diagnostic tests has become an important goal of health care reform. We conducted the present study to determine the incidence and nature of abnormalities on neck CT and barium esophagram examinations performed in the work-up of patients with isolated GS and to assess which imaging technique contributed most effectively to the clinical management of these patients.     

A biomechanical comparison of three lower extremity tendons for ligamentous reconstruction about the knee

Purpose: The purpose of this investigation was to evaluate 3 previously unreported allograft tendons for use in knee surgery. These are the doubled tibialis anterior (TA), doubled tibialis posterior (TP), and doubled peroneus longus (PL) tendons. Type of Study: A biomechanical evaluation of the properties of the TA, TP, and PL. Methods: Sixteen fresh-frozen cadaveric lower limbs were used for testing. All specimens had the TA, TP, and PL tendons harvested. All specimens were tested in a custom-designed hydraulic testing machine using dry ice clamps. Each tendon was elongated at a rate of 1 mm/s. Load and displacement were recorded with an analog to digital interface board. Stiffness, modulus of elasticity, and stress and strain at failure were calculated. Results: The average tested lengths of the TA, TP, and PL were 37 cm (range, 13–68 cm), 33 cm (range, 7–74 cm), and 42 cm (range, 17–69 cm), respectively. The average cross-sectional areas of the doubled TA, TP, and PL were 38 mm², 48 mm², and 37 mm², respectively. The average failure loads for the doubled TA, TP, and PL tendons were 3,412 N, 3,391 N, and 2,483 N, respectively. The maximum stresses of the 3 tendons did not differ significantly (85–108 Mpa). The TA had the greatest stiffness (344 N/mm), followed by the TP (302 N/mm) and the PL (244 N/mm). Previous authors have documented the biomechanical strength of grafts for ACL reconstruction between 1,700 and 2,900 Newtons. The ultimate tensile strength and stiffness reported for the TA and TP grafts exceeded that for all previously reported grafts, including the doubled semitendinosus-gracilis. Conclusions: The TA, TP, and PL tendons showed excellent biomechanical properties when compared with historical data evaluating other graft sources. The biomechanical properties observed for the TA, TP, and PL were noted in specimens despite an average age of 78.3 years.     

ST6Gal-I expression in ovarian cancer cells promotes an invasive phenotype by altering integrin glycosylation and function

Background

Ovarian adenocarcinoma is not generally discovered in patients until there has been widespread intraperitoneal dissemination, which is why ovarian cancer is the deadliest gynecologic malignancy. Though incompletely understood, the mechanism of peritoneal metastasis relies on primary tumor cells being able to detach themselves from the tumor, escape normal apoptotic pathways while free floating, and adhere to, and eventually invade through, the peritoneal surface. Our laboratory has previously shown that the Golgi glycosyltransferase, ST6Gal-I, mediates the hypersialylation of β₁ integrins in colon adenocarcinoma, which leads to a more metastatic tumor cell phenotype. Interestingly, ST6Gal-I mRNA is known to be upregulated in metastatic ovarian cancer, therefore the goal of the present study was to determine whether ST6Gal-I confers a similarly aggressive phenotype to ovarian tumor cells.

Methods

Three ovarian carcinoma cell lines were screened for ST6Gal-I expression, and two of these, PA-1 and SKOV3, were found to produce ST6Gal-I protein. The third cell line, OV4, lacked endogenous ST6Gal-I. In order to understand the effects of ST6Gal-I on cell behavior, OV4 cells were stably-transduced with ST6Gal-I using a lentiviral vector, and integrin-mediated responses were compared in parental and ST6Gal-I-expressing cells.

Results

Forced expression of ST6Gal-I in OV4 cells, resulting in sialylation of β1 integrins, induced greater cell adhesion to, and migration toward, collagen I. Similarly, ST6Gal-I expressing cells were more invasive through Matrigel.

Conclusion

ST6Gal-I mediated sialylation of β1 integrins in ovarian cancer cells may contribute to peritoneal metastasis by altering tumor cell adhesion and migration through extracellular matrix.     

De novo mapping of α-helix recognition sites on protein surfaces using unbiased libraries

The α-helix is one of the most common protein surface recognition motifs found in nature, and its unique amide-cloaking properties also enable α-helical polypeptide motifs to exist in membranes. Together, these properties have inspired the development of α-helically constrained (Helicon) therapeutics that can enter cells and bind targets that have been considered “undruggable”, such as protein–protein interactions. To date, no general method for discovering α-helical binders to proteins has been reported, limiting Helicon drug discovery to only those proteins with previously characterized α-helix recognition sites, and restricting the starting chemical matter to those known α-helical binders. Here, we report a general and rapid screening method to empirically map the α-helix binding sites on a broad range of target proteins in parallel using large, unbiased Helicon phage display libraries and next-generation sequencing. We apply this method to screen six structurally diverse protein domains, only one of which had been previously reported to bind isolated α-helical peptides, discovering 20 families that collectively comprise several hundred individual Helicons. Analysis of 14 X-ray cocrystal structures reveals at least nine distinct α-helix recognition sites across these six proteins, and biochemical and biophysical studies show that these Helicons can block protein–protein interactions, inhibit enzymatic activity, induce conformational rearrangements, and cause protein dimerization. We anticipate that this method will prove broadly useful for the study of protein recognition and for the development of both biochemical tools and therapeutics for traditionally challenging protein targets.

Recent advances in identifying human disease targets have not been matched by advances in the ability to drug these targets. This actionability gap is largely due to the fact that neither of the two main classes of approved therapeutics – biologics and small molecules – can simultaneously address target accessibility and selective target engagement. Biologics, despite an impressive ability to engage diverse target proteins, are largely restricted to an extracellular operating theater, as their size and polarity render them unable to cross biological membranes. Small molecules, in contrast, can access the intracellular space, but cannot bind with high affinity and specificity to the vast majority of proteins that are found there (1).This disconnect between the ability to identify disease targets and the ability to drug them with high strength and specificity has created an impetus to develop new classes of drugs – ones that can engage intracellular proteins that lack the deep hydrophobic pocket ordinarily required for small-molecule binding. In nature, such “undruggable” proteins are often targeted with macrocyclic molecules, frequently peptidic in structure, whose large size compared with small molecules enables them to bind with high affinity and specificity to protein surfaces.Significant efforts have been made to elucidate the mechanisms of cell entry for these natural products, which possess molecular weights of 700 to 1,200 Da or higher, well beyond the typical range for cell penetration in small-molecule drug discovery (2). While the mechanisms of cell entry are complex and vary from molecule to molecule, a substantial body of research on peptidic macrocycles has highlighted the importance of desolvating amide protons and reducing their exposure to the membrane interior as a key driver in passive, thermal diffusion across the lipid bilayer (2, 3) – a phenomenon we refer to as amide-proton cloaking. The amide proton, present between every residue in a polypeptide chain, is highly electropositive and forms a strong hydrogen-bonding interaction with water. This poses a substantial hurdle for membrane permeability, since tightly bound solvent water molecules must be shed prior to entering the lipid bilayer. Exposed amide groups incur a further energetic penalty upon membrane entry due to unfavorable electrostatic interactions with the low-dielectric environment of the membrane interior. Consequently, most peptides and proteins are unable to cross membranes.For peptide macrocycles that are able to permeate the membrane, these problematic amide protons are typically removed either by replacing the amide with an ester, replacing it with a methyl group, or cloaking it from solvent water through the formation of intramolecular hydrogen bonds between the amide proton groups and a hydrogen bond-accepting group elsewhere in the molecule, often a carbonyl. Indeed, the paradigmatic example of a natural peptide macrocycle that exhibits robust cytosolic exposure, cyclosporine A (CsA), employs both N-methylation and cloaking through transannular hydrogen bonding (4). Extensive work by several research groups has shown that these strategies can be applied as design principles to endow artificial macrocycles with the ability to cross membranes (5–7).In the context of folded proteins, nature has offered an alternative structural solution to the problem of amide proton cloaking: the α-helix, a protein secondary structure that is defined by repeating intramolecular hydrogen bonds between the amide proton group of one residue and the carbonyl of the amino acid located four residues N terminal to it. The intrinsic ability of α-helices to cloak their own amide protons explains their widespread prevalence in natural transmembrane proteins (8). Nuclear-encoded transmembrane proteins in eukaryotes are almost exclusively α-helical, and the only alternative transmembrane fold found in nature is the bacterially derived β-barrel, a helical structure that also cloaks amide protons via an intramolecular hydrogen bonding network, albeit in a significantly larger structure than single α-helices that is impractical for the development of synthetic drugs.Just as CsA has served as the inspiration for the design of mimetic head-to-tail cyclized peptide ligands, so have proteinaceous α-helices inspired efforts to recapitulate nature’s design features in small, synthetic, α-helically constrained peptides (Helicons) that are hyperstabilized through the incorporation of a structural brace, also known as a “staple” (9–12). One of these, the all-hydrocarbon staple formed by ring-closing metathesis, has been extensively studied and is the basis for a drug candidate that targets the challenging proteins MDM2 and MDMX, currently undergoing Phase II clinical trials (13, 14).Rational design of Helicons is difficult given the inability to systematically define the α-helix binding sites on a protein’s surface, and to identify Helicons that bind to those sites. This limitation has restricted research on Helicons to only those protein targets for which naturally occurring or previously characterized α-helical binders were known, with the Helicons generated from fragments of the known binders (3). Here, we report a rapid, high-throughput screening platform utilizing phage display that enables an unbiased mapping of the α-helical interactome of a given protein without any prior knowledge of its structure or known binding partners. We show that this platform is capable of identifying α-helix binding sites on the surfaces of a range of protein folds, including many for which no α-helical binders are known to exist. Helicons that bind these sites are able to impact diverse protein functions, including inhibiting protein–protein interactions, inhibiting enzymatic activity, inducing dimerization, and inducing conformational changes. Analysis of 14 high-resolution crystal structures of Helicon–protein complexes across six different protein domains reveals a range of binding modes, all of which are “side-on”, i.e., mediated exclusively by Helicon side-chains rather than involving main chain amide interactions. This screening platform significantly expands the universe of proteins that can be bound by Helicons, and furthers the pursuit of targeting undruggable proteins.