Design and investigation of interactions of novel peptide conjugates of purine and pyrimidine derivatives with EGFR and its mutant T790M/L858R: an in silico and laboratory study.

Peptide-based therapeutics have been gaining attention due to their ability to actively target tumor cells. Additionally, several varieties of nucleotide derivatives have been developed to reduce cell proliferation and induce apoptosis of tumor cells. In this work, we have developed novel peptide conjugates with newly designed purine analogs and pyrimidine derivatives and explored the binding interactions with the kinase domain of wild-type EGFR and its mutant EGFR [L858R/ T790M] which are known to be over-expressed in tumor cells. The peptides explored included WNWKV (derived from sea cucumber) and LARFFS, which in previous work was predicted to bind to Domain I of EGFR. Computational studies conducted to explore binding interactions include molecular docking studies, molecular dynamics simulations and MMGBSA to investigate the binding abilities and stability of the complexes. The results indicate that conjugation enhanced binding capabilities, particularly for the WNWKV conjugates. MMGBSA analysis revealed nearly twofold higher binding toward the T790M/L858R double mutant receptor. Several conjugates were shown to have strong and stable binding with both wild-type and mutant EGFR. As a proof of concept, we synthesized pyrimidine conjugates with both peptides and determined the KD values using SPR analysis. The results corroborated with the computational analyses. Additionally, cell viability and apoptosis studies with lung cancer cells expressing the wild-type and double mutant proteins revealed that the WNWKV conjugate showed greater potency than the LARFFS conjugate, while LARFFS peptide alone showed poor binding to the kinase domain. Thus, we have designed peptide conjugates that show potential for further laboratory studies for developing therapeutics for targeting the EGFR receptor and its mutant T790M/L858R.

Deep Brain Stimulation for Chronic Cluster Headaches: A Systematic Review and Meta-Analysis.

BACKGROUND: Chronic cluster headache (CCH) is a severe and debilitating sub-type of trigeminal autonomic cephalalgia that can be resistant to medical management and associated with significant impairment in quality of life. Studies of deep brain stimulation (DBS) for CCH have provided promising results but have not been assessed in a comprehensive systematic review/meta-analysis. OBJECTIVE: The objective was to perform a systematic literature review and meta-analysis of patients with CCH treated with DBS to provide insight on safety and efficacy. METHODS: A systematic review and meta-analysis were performed according to PRISMA 2020 guidelines. 16 studies were included in final analysis. A random-effects model was used to meta-analyze data. RESULTS: Sixteen studies reported 108 cases for data extraction and analysis. DBS was feasible in >99% of cases and was performed either awake or asleep. Meta-analysis revealed that the mean difference in headache attack frequency and headache intensity after DBS were statistically significant (p < 0.0001). Utilization of microelectrode recording was associated with statistically significant improvement in headache intensity postoperatively (p = 0.006). The average overall follow-up period was 45.4 months and ranged from 1 to 144 months. Death occurred in <1%. The rate of major complications was 16.67%. CONCLUSIONS: DBS for CCHs is a feasible surgical technique with a reasonable safety profile that can be successfully performed either awake or asleep. In carefully selected patients, approximately 70% of patients achieve excellent control of their headaches.

Interactions of Nanoscale Self-Assembled Peptide-Based Assemblies with Glioblastoma Cell Models and Spheroids.

Peptide nanoassemblies have garnered remarkable importance in the development of novel nanoscale biomaterials for drug delivery into tumor cells. Taking advantage of receptor mediated recognition of two known peptides, angiopep-2 (TFFYGGSRGKRNNFKTEEY) and A-COOP-K (ACGLSGLC10 VAK) that bind to the over-expressed receptors low density lipoprotein (LRP-1) and fatty acid binding protein (FABP3) respectively, we have developed new peptide conjugates by combining the anti-inflammatory, antitumor compound azelaic acid with angiopep-2, which efficiently self-assembled into nanofibers. Those nanofibers were then functionalized with the A-COOP-K sequence and formed supramolecular hierarchical structures that were found to entrap the chemotherapeutic drug doxorubicin efficaciously. Furthermore, the nanoassemblies were found to release the drug in a dose-dependent manner and showed a stepwise increase over a period of 2 weeks under acidic conditions. Two cell lines (U-87-MG and U-138-MG) were utilized as models for glioblastoma cells grown in the presence of serum and under serum-free conditions to mimic the growth conditions of natural tumors. The drug entrapped assemblies were found to inhibit the cell proliferation of both U-87 and U-138MG glioblastoma cells. Three dimensional spheroids of different sizes were grown to mimic the tumors and evaluate the efficacy of drug release and internalization. Our results indicated that the nanoassemblies were found to have higher internalization of DOX and were well-spread throughout the spheroids grown, particularly under serum-free conditions. The nanoassemblies also displayed blood-brain barrier penetration when tested with a multicellular in vitro model. Such self-assembled nanostructures with targeting ability may provide a suitable platform for the development of new peptide-based biomaterials that can provide more insights about the mechanistic approach for drug delivery for not only 2D cell cultures but also 3D tumoroids that mimic the tumor microenvironments.

An investigation of binding interactions of tumor-targeted peptide conjugated polyphenols with the kinase domain of ephrin B4 and B2 receptors.

Recent studies have shown that Ephrin receptors may be upregulated in several types of cancers including breast, ovarian and endometrial cancers, making them a target for drug design. In this work, we have utilized a target-hopping approach to design new natural product-peptide conjugates and examined their interactions with the kinase-binding domain of EphB4 and EphB2 receptors. The peptide sequences were generated through point mutations of the known EphB4 antagonist peptide TNYLFSPNGPIA. Their anticancer properties and secondary structures were analyzed computationally. Conjugates of most optimum of peptides were then designed by binding the N-terminal of the peptides with the free carboxyl group of the polyphenols sinapate, gallate and coumarate, which are known for their inherent anticancer properties. To investigate if these conjugates have a potential to bind to the kinase domain, we carried out docking studies and MMGBSA free energy calculations of the trajectories based on the molecular dynamics simulations, with both the apo and the ATP bound kinase domains of both receptors. In most cases binding interactions occurred within the catalytic loop region, while in some cases the conjugates were found to spread out across the N-lobe and the DFG motif region. The conjugates were further tested for prediction of pharmacokinetic properties using ADME studies. Our results indicated that the conjugates were lipophilic and MDCK permeable with no CYP interactions. These findings provide an insight into the molecular interactions of these peptides and conjugates with the kinase domain of the EphB4 and EphB2 receptor. As a proof of concept, we synthesized and carried out SPR analysis with two of the conjugates (gallate-TNYLFSPNGPIA and sinapate-TNYLFSPNGPIA). Results indicated that the conjugates showed higher binding with the EphB4 receptor and minimal binding to EphB2 receptor. Sinapate-TNYLFSPNGPIA showed inhibitory activity against EphB4. These studies reveal that some of the conjugates may be developed for further investigation into in vitro and in vivo studies and potential development as therapeutics.

The Sensory Auricular Branch of the Facial Nerve and its Relationship to Landmarks of the Facial Recess.

OBJECTIVE: Delineate the anatomic relationship of the sensory auricular branch (SAB) of the facial nerve to other structures of the facial recess. METHODS: Ten adult cadaveric temporal bones were randomly selected and dissected under operative microscopy. Linear and angular measurements were obtained for the following parameters: (1) the distance from the tip of the short process of the incus to the point of convergence of the SAB and the main trunk of the facial nerve; (2) the distance from the point of convergence of the SAB and the main trunk of the facial nerve to the chorda tympani (CT) division from the main trunk; (3) the distance from the bifurcation of the CT and facial nerve to the crossover point of the SAB/CT; (4) the angle at which the SAB merges with the main trunk (Y°), and (5) the angle at which the CT divides off the main trunk (X°). RESULTS: The mean distance from the tip of the short process of the incus to the SAB takeoff was 8.7 ± 1.83 mm (range 6-13 mm). The mean distance from the SAB to the CT division from the main trunk was 5.9 ± 2.41 mm (range 3-10 mm). The mean angle at which the SAB merged with the main trunk of the facial nerve was 38.5 ± 12.63° (range 25°-68°). The mean CT-main trunk angle was 16 ± 4.24° (range 8°-21°). The branching point of the SAB from the facial nerve approximately bisected the facial recess. CONCLUSION: Recognizing the SAB and knowing its relationships to surrounding anatomy provides a useful adjunctive landmark for the identification of the main trunk of the facial nerve's mastoid segment. LEVEL OF EVIDENCE: 4.

Pott's puffy tumor: A comprehensive review of the literature.

PURPOSE: Pott's puffy tumor (PPT) is a rare clinical entity characterized by osteomyelitis of the frontal bone with subperiosteal abscess collection. The frequency of reported cases of PPT in the literature has increased in recent years. Previous reviews of PPT exist primarily in the form of small, retrospective case series and anecdotal case reports. Therefore, the aim of this study is to provide the literature's largest comprehensive, up-to-date review of the essential clinical findings, diagnostic modalities, microbiologic considerations, and treatment approaches utilized in the management of PPT, both in pediatric and adult populations. MATERIALS AND METHODS: We searched MEDLINE, PubMed, and Embase databases for English-language studies published from January 1950 through January 30, 2022. The authors reviewed all cases of PPT, focusing specifically on those describing therapeutic management of PPT. A total of 321 patients were included, consisting of 318 patients (from 216 articles) and an additional 3 adult cases from our institution. RESULTS: PPT most often results from untreated rhinosinusitis, as well as direct head trauma, substance use, and odontogenic disease. Infections are classically polymicrobial with an anaerobe-predominant microbiome. Both CT and MRI imaging modalities are commonly obtained for presurgical assessment of sinusitis and intracranial extension. The core of treatment is an early and aggressive approach to prevent long-term complications. A significant association exists between surgical management and clinical outcomes for patients with PPT. Recent literature suggests endoscopic sinus surgery is essential for successful disease resolution. CONCLUSIONS: PPT is an important and relatively morbid disease process that is often underrecognized and misdiagnosed at presentation due to its variable clinical presentation. Management of PPT includes both antimicrobial therapy and surgical intervention. Determination of the optimal approach depends on patient clinical features including age, history of prior endoscopic sinus surgery, and presence of intracranial involvement on presentation. An individualized, targeted, and interdisciplinary approach to the treatment of PPT is critical for successful disease resolution.

Evaluation of real-world referential and probabilistic patient matching to advance patient identification strategy.

OBJECTIVE: This study sought both to support evidence-based patient identity policy development by illustrating an approach for formally evaluating operational matching methods, and also to characterize the performance of both referential and probabilistic patient matching algorithms using real-world demographic data. MATERIALS AND METHODS: We assessed matching accuracy for referential and probabilistic matching algorithms using a manually reviewed 30 000 record gold standard reference dataset derived from a large health information exchange containing over 47 million patient registrations. We applied referential and probabilistic algorithms to this dataset and compared the outputs to the gold standard. We computed performance metrics including sensitivity (recall), positive predictive value (precision), and F-score for each algorithm. RESULTS: The probabilistic algorithm exhibited sensitivity, positive predictive value (PPV), and F-score of .6366, 0.9995, and 0.7778, respectively. The referential algorithm exhibited corresponding sensitivity, PPV, and F-score values of 0.9351, 0.9996, and 0.9663, respectively. Treating discordant and limited-data records as nonmatches increased referential match sensitivity to 0.9578. Compared to the more traditional probabilistic approach, referential matching exhibits greater accuracy. CONCLUSIONS: Referential patient matching, an increasingly popular method among health IT vendors, demonstrated notably greater accuracy than a more traditional probabilistic approach without the adaptation of the algorithm to the data that the traditional probabilistic approach usually requires. Health IT policymakers, including the Office of the National Coordinator for Health Information Technology (ONC), should explore strategies to expand the evidence base for real-world matching system performance, given the need for an evidence-based patient identity strategy.

Retrospective analysis of post-tracheostomy complications.

OBJECTIVE: To elucidate patient, disease, and surgical factors that are significantly associated with 90-day tracheostomy complications, readmissions, and mortality. STUDY DESIGN: Retrospective case series with chart review. SETTING: A single academic tertiary care center between 2011 and 2018. METHODS: Patients who underwent tracheostomy by any technique for any indication were included. Demographic, disease, and operative details were examined. Multivariable analysis was performed to determine factors associated with 90-day complications, 90-day readmissions, and overall mortality. RESULTS: 697 patients were included. 75% of patients had severe comorbidity (ACE-27 score of 3).1 Patients were intubated for 12 days prior to tracheostomy placement on average. The primary indication was ventilator dependence due to critical illness (85%). 74% were performed open and 26% percutaneous. 10% of patients had a tracheostomy-related complication within 90 days. Complications occurred at a median of post-operative day 11, and hemorrhage was most common (n = 35). 14 patients required immediate return to the operating room, and 3 patients died of their complication, all within 3 days of tracheostomy placement. 40% of patients undergoing tracheostomy died within 30 days. In multivariable analysis, only a documented difficult tracheostomy placement was significantly associated with a 90-day complication. CONCLUSIONS: While complications after tracheostomy are infrequent, they are often severe. A heightened level of preparedness to immediately manage accidental tracheostomy decannulation or hemorrhage is required for patients with a difficult tracheostomy placement. 30-day mortality is high, which reinforces the need for multi-disciplinary evaluation, including palliative care, to determine appropriate candidacy for tracheostomy.

BRAF in Lung Cancers: Analysis of Patient Cases Reveals Recurrent BRAF Mutations, Fusions, Kinase Duplications, and Concurrent Alterations.

PURPOSE: Dabrafenib and trametinib are approved for the management of advanced non-small-cell lung cancers (NSCLCs) that harbor BRAF V600E mutations. Small series and pan-cancer analyses have identified non-V600 alterations as therapeutic targets. We sought to examine a large genomic data set to comprehensively characterize non-V600 BRAF alterations in lung cancer. PATIENTS AND METHODS: A total of 23,396 patients with lung cancer provided data to assay with comprehensive genomic profiling. Data were reviewed for predicted pathogenic BRAF base substitutions, short insertions and deletions, copy number changes, and rearrangements. RESULTS: Adenocarcinomas represented 65% of the occurrences; NSCLC not otherwise specified (NOS), 15%; squamous cell carcinoma, 12%; and small-cell lung carcinoma, 5%. BRAF was altered in 4.5% (1,048 of 23,396) of all tumors; 37.4% (n = 397) were BRAF V600E, 38% were BRAF non-V600E activating mutations, and 18% were BRAF inactivating. Rearrangements were observed at a frequency of 4.3% and consisted of N-terminal deletions (NTDs; 0.75%), kinase domain duplications (KDDs; 0.75%), and BRAF fusions (2.8%). The fusions involved three recurrent fusion partners: ARMC10, DOCK4, and TRIM24. BRAF V600E was associated with co-occurrence of SETD2 alterations, but other BRAF alterations were not and were instead associated with CDKN2A, TP53, and STK11 alterations (P < .05). Potential mechanisms of acquired resistance to BRAF V600E inhibition are demonstrated. CONCLUSION: This series characterized the frequent occurrence (4.4%) of BRAF alterations in lung cancers. Recurrent BRAF alterations in NSCLC adenocarcinoma are comparable to the frequency of other NSCLC oncogenic drivers, such as ALK, and exceed that of ROS1 or RET. This work supports a broad profiling approach in lung cancers and suggests that non-V600E BRAF alterations represent a subgroup of lung cancers in which targeted therapy should be considered.

Profiling of 149 Salivary Duct Carcinomas, Carcinoma Ex Pleomorphic Adenomas, and Adenocarcinomas, Not Otherwise Specified Reveals Actionable Genomic Alterations.

PURPOSE: We sought to identify genomic alterations (GA) in salivary gland adenocarcinomas, not otherwise specified (NOS), salivary duct carcinomas (SDC), carcinoma ex pleomorphic adenoma (ca ex PA), and salivary carcinoma, NOS. EXPERIMENTAL DESIGN: DNA was extracted from 149 tumors. Comprehensive genomic profiling (CGP) was performed on hybridization-captured adaptor ligation-based libraries of 182 or 315 cancer-related genes plus introns from 14 or 28 genes frequently rearranged for cancer and evaluated for all classes of GAs. RESULTS: A total of 590 GAs were found in 157 unique genes (mean 3.9/tumor). GAs in the PI3K/AKT/mTOR pathway were more common in SDC (53.6%) than other histologies (P = 0.019) Cyclin-dependent kinase GAs varied among all histotypes: adenocarcinoma, NOS (34.6%); SDC (12.2%); ca ex PA (16.7%); carcinoma, NOS (31.2%; P = 0.043). RAS GAs were observed: adenocarcinoma, NOS (17.3%); SDC (26.8%); ca ex PA (4.2%); and carcinoma, NOS (9.4%; P = 0.054). ERBB2 GAs, including amplifications and mutations, were common: adenocarcinoma, NOS (13.5%); SDC (26.8%); ca ex PA (29.2%); carcinoma, NOS (18.8; P = 0.249). Other notable GAs include TP53 in >45% of each histotype; NOTCH1: adenocarcinoma, NOS (7.7%), ca ex PA (8.3%), carcinoma, NOS (21.6%); NF1: adenocarcinoma, NOS (9.6%), SDC (17.1%), carcinoma, NOS (18.8%). RET fusions were identified in one adenocarcinoma, NOS (CCDC6-RET) and two SDCs (NCOA4-RET). Clinical responses were observed in patients treated with anti-HER2 and anti-RET-targeted therapies. CONCLUSIONS: CGP of salivary adenocarcinoma, NOS, SDCs, ca ex PA, and carcinoma, NOS revealed diverse GAs that may lead to novel treatment options. Clin Cancer Res; 22(24); 6061-8. ©2016 AACR.

SFRP1 reduction results in an increased sensitivity to TGF-ß signaling.

BACKGROUND: Transforming growth factor (TGF)-ß plays a dual role during mammary gland development and tumorigenesis and has been shown to stimulate epithelial-mesenchymal transition (EMT) as well as cellular migration. The Wnt/ß-catenin pathway is also implicated in EMT and inappropriate activation of the Wnt/ß-catenin signaling pathway leads to the development of several human cancers, including breast cancer. Secreted frizzled-related protein 1 (SFRP1) antagonizes this pathway and loss of SFRP1 expression is frequently observed in breast tumors and breast cancer cell lines. We previously showed that when SFRP1 is knocked down in immortalized non-malignant mammary epithelial cells, the cells (TERT-siSFRP1) acquire characteristics associated with breast tumor initiating cells. The phenotypic and genotypic changes that occur in response to SFRP1 loss are consistent with EMT, including a substantial increase in the expression of ZEB2. Considering that ZEB2 has been shown to interact with mediators of TGF-ß signaling, we sought to determine whether TGF-ß signaling is altered in TERT-siSFRP1 cells. METHODS: Luciferase reporter assays and real-time PCR analysis were employed to measure TGF-ß transcriptional targets. Western blot analysis was used to evaluate TGF-ß-mediated ERK1/2 phosphorylation. Migration chamber assays were utilized to quantify cellular migration. TERT-siSFRP1 cells were transfected with Stealth RNAi™ siRNA in order to knock-down the expression of ZEB2. RESULTS: TERT-siSFRP1 cells exhibit a significant increase in both TGF-ß-mediated luciferase activity as well as TGF-ß transcriptional targets, including Integrin ß3 and PAI-1. Phosphorylation of ERK1/2 is increased in TERT-siSFRP1 cells in response to enhanced TGF-ß signaling. Furthermore, when the TGF-ß pathway is blocked with a TGF-ßR antagonist (LY364947), cellular migration is significantly hindered. Finally, we found that when ZEB2 is knocked-down, there is a significant reduction in the expression of exogeneous and endogenous TGF-ß transcriptional targets and cellular migration is impeded. CONCLUSIONS: We demonstrate that down-regulation of SFRP1 renders mammary epithelial cells more sensitive to TGF-ß signaling which can be partially ameliorated by blocking the expression of ZEB2.