Supporting information for: "Nortriptyline Hydrochloride Solubility-pH Profiles in a Saline Phosphate Buffer: Drug-Phosphate Complexes and Multiple pHmax Domains with a Gibbs Phase Rule “Soft” Constraints"

Additional experimental details; preparation of stock suspensions for low-to-high pH and high-to-low pH titrations; titration and solubility data for titration sets 1–11; elemental analysis data from titration sets 9, 3, 6, and 7; and HPLC UV/VIS analysis─sample chromatograms and calibration diagramThe supporting information for: Marković OS, Patel NG, Serajuddin ATM, Avdeef A, Verbić TŽ. Nortriptyline Hydrochloride Solubility-pH Profiles in a Saline Phosphate Buffer: Drug-Phosphate Complexes and Multiple pHmax Domains with a Gibbs Phase Rule “Soft” Constraints. in Molecular Pharmaceutics. 2022;19(2):710-719. doi: [10.1021/acs.molpharmaceut.1c00919]Published manuscript: [https://cer.ihtm.bg.ac.rs/handle/123456789/4934]The peer-reviewed version: [https://cer.ihtm.bg.ac.rs/handle/123456789/4936

pH-Dependent solubility profile of nortriptyline hydrochloride

Solubility is important physicochemical parameter and determines drug stability, bioavailability and therapeutic action. The aim of this study was to examine solubility of nortriptyline hydrochloride in a wide pH range, using pH-Ramp Shake-Flask method, already applied to desipramine hydrochloride [1] and based of recently published recommendations [2]. Solubility was measured in phosphate buffer, in chloride-free media and phoshate-free media, using both nortriptyline base and nortriptyline hydrochloride as starting material. Elemental analysis, termogravimetric analysis, differential scaning calorimetric analysis and powder X-ray diffraction analysis were used for solid precipitate analysis.Rastvorljivost je značajno fizičko-hemijsko svojstvo biološki aktivnih i potencijalno biološki aktivnih supstancija, koje određuje stabilnost, biodostupnost i terapeutsko dejstvo leka. Cilj ovog rada je ispitivanje rastvorljivosti nortriptilin-hidrohlorida, pomoću pH-Ramp Shake-Flask metode, prethodno primenjene na desipramin-hidrohlorid [1]. Eksperimenti su izvedeni prema novim preporukama iz literature [2]. Rastvorljivost je određena u fosfatnom puferu, u sistemu bez hlorida i sistemu bez fosfata, koristeći nortriptilin bazu i nortriptilin-hidrohlorid. Urađena je i katakterizacija čvrste faze pomoću elementalne analize, termogravimetrije, diferencijalne skenirajuće kalorimetrije i difrakcije X-zraka.57th MEETING OF THE SERBIAN CHEMICAL SOCIETY, Kragujevac, Serbia, June 18-19, 202

Identification of Human Papillomavirus Infection in Cancer Tissue by Targeted Next-generation Sequencing

Human papillomaviruses (HPV) are oncogenic DNA viruses implicated in squamous cell carcinomas of several anatomic sites, as well as endocervical adenocarcinomas. Identification of HPV is an actionable finding in some carcinomas, potentially influencing tumor classification, prognosis, and management. We incorporated capture probes for oncogenic HPV strains 16 and 18 into a broader next-generation sequencing (NGS) panel designed to identify actionable mutations in solid malignancies. A total of 21 head and neck, genitourinary and gynecological squamous cell carcinomas and endocervical adenocarcinomas were sequenced as part of the UNCSeq project. Using p16 immunohistochemical results as the gold standard, we set a cutoff for proportion of aligned HPV reads that maximized performance of our NGS assay (92% sensitive, 100% specific for HPV). These results suggest that sequencing of oncogenic pathogens can be incorporated into targeted NGS panels, extending the clinical utility of genomic assays

Germline Analysis from Tumor–Germline Sequencing Dyads to Identify Clinically Actionable Secondary Findings

To evaluate germline variants in hereditary cancer susceptibility genes among unselected cancer patients undergoing tumor-germline sequencing

Multi-Institutional FASTQ File Exchange as a Means of Proficiency Testing for Next-Generation Sequencing Bioinformatics and Variant Interpretation

Next-generation sequencing is becoming increasingly common in clinical laboratories worldwide and is revolutionizing clinical molecular testing. However, the large amounts of raw data produced by next-generation sequencing assays and the need for complex bioinformatics analyses present unique challenges. Proficiency testing in clinical laboratories has traditionally been designed to evaluate assays in their entirety; however, it can be alternatively applied to separate assay components. We developed and implemented a multi-institutional proficiency testing approach to directly assess custom bioinformatics and variant interpretation processes. Six clinical laboratories, all of which use the same commercial library preparation kit for next-generation sequencing analysis of tumor specimens, each submitted raw data (FASTQ files) from four samples. These 24 file sets were then deidentified and redistributed to five of the institutions for analysis and interpretation according to their clinically validated approach. Among the laboratories, there was a high rate of concordance in the calling of single-nucleotide variants, in particular those we considered clinically significant (100% concordance). However, there was significant discordance in the calling of clinically significant insertions/deletions, with only two of seven being called by all participating laboratories. Missed calls were addressed by each laboratory to improve their bioinformatics processes. Thus, through our alternative proficiency testing approach, we identified the bioinformatic detection of insertions/deletions as an area of particular concern for clinical laboratories performing next-generation sequencing testing

Metabolic and Functional Genomic Studies Identify Deoxythymidylate Kinase as a Target in LKB1-Mutant Lung Cancer

The LKB1/STK11 tumor suppressor encodes a serine/threonine kinase which coordinates cell growth, polarity, motility, and metabolism. In non-small cell lung cancer, LKB1 is somatically inactivated in 25-30% of cases, often concurrently with activating KRAS mutation. Here, we employed an integrative approach to define novel therapeutic targets in KRAS-driven LKB1 mutant lung cancers. High-throughput RNAi screens in lung cancer cell lines from genetically engineered mouse models driven by activated KRAS with or without coincident Lkb1 deletion led to the identification of Dtymk, encoding deoxythymidylate kinase which catalyzes dTTP biosynthesis, as synthetically lethal with Lkb1 deficiency in mouse and human lung cancer lines. Global metabolite profiling demonstrated that Lkb1-null cells had striking decreases in multiple nucleotide metabolites as compared to the Lkb1-wt cells. Thus, LKB1 mutant lung cancers have deficits in nucleotide metabolism conferring hypersensitivity to DTYMK inhibition, suggesting that DTYMK is a potential therapeutic target in this aggressive subset of tumors

Diagnostic accuracy of The Bethesda system for reporting thyroid cytopathology: A Five years Study

Introduction: Thyroid nodules are a common clinical problem. It is important to differentiate benign from malignant nodules. Fine needle aspiration is utilized as a preoperative diagnostic technique which is safe, simple and cost effective for triaging patients with thyroid nodules. Methods: The study was conducted at Histopathology department of B.J Medical college, Ahmedabad. It involved period of 5 years patients who presented with thyroid swelling and underwent FNAC and Histopathology. Out of these 1325 cases, 210 patients subsequently underwent surgical excision. Results of final histopathology were correlated with cytologic diagnosis. Results: Histopathologic correlation was done in 210 cases. Out of total 210 cases, 3 cases were diagnosed as Non diagnostic or unsatisfactory,199 cases were diagnosed as Bethesda II and No case was Bethesda III while 4 cases were categorized Bethesda IV and 1 case were Bethesda V and 3 cases were Bethesda VI. The incidence of malignancy in Bethesda categories through were 0%,7.53%, 0%,100%,100% and 100% respectively. Overall accuracy Of FNA cytology was 93.8% with 81.25 % sensitivity and 96.06 % specificity. Conclusion: Our study validated the accuracy of TBSRTC in our setup which is concordant with other studies. Therefore, we recommend routine use of TBSRTC for reporting thyroid cytopathology for initial workup. However, risk of malignancy was found to be significantly high in Bethesda IV, V and VI category to warrant further workup including ultrasound/thyroid scan in addition to repeat FNAC