Analysis of odontoblast gene expression using a novel approach,laser capture microdissection

Studying the mechanisms of molecular interactions in developing tissues demands sensitive molecular biological in vivo and in vitro techniques. Laser capture microdissection (LCM) allows for the isolation of mRNA in histological sections even from single cells, thus enabling the identification of in vivo gene expression products in closely circumscribed tissue areas. The aims of this study were to assess the optimal fixation, processing, and staining conditions to retrieve RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of alpha 1(I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA isolated from day 0 and day 3 captured odontoblasts was sufficient in quantity and quality. Our results indicate that individual odontoblasts obtained by LCM are morphologically intact and chemically unaltered, allowing accurate molecular and biochemical analyses.     

Expression of sodium channel α- and β-subunits in the nervous system of themyelin-deficient rat

Summary Using subtype-specific riboprobes and a non-isotopein situ hybridization technique, the pattern of expression of the mRNAs for voltage dependent sodium channel -subunits I, II, III and NaG, and the ₁-subumt were compared inmyelin-deficient rats and unaffected male littermates. Tissues examined included the hippocampus, cerebellum, spinal cord and dorsal root ganglia. Previous studies have demonstrated that the expression of sodium channel - and ₁-subunits follows a distinct temporal and spatial pattern during development, characterized in part by greater expression of -subunit III and its mRNA during development than in the adult. We examined animals of 20–22 days of age, a time when, according to earlier reports, the unaffected animals should nearly have reached an adult expression pattern. Normal male littermates were indeed found to express a sodium channel subunit mRNA pattern generally consistent with previous reports on adult rats.Myelin-deficient animals exhibited an expression pattern identical to the unaffected littermates, indicating that myelination is not required for the progression from the embryonic to the adult expression pattern of sodium channel subunits.     

Laparoscopic management of suspicious adnexal masses

OBJECTIVE: Our aim was to evaluate the feasibility and applicability of operative laparoscopy in the management of adnexal masses that do not meet the standard serum CA 125 and ultrasonographic criteria for benignity. STUDY DESIGN: One hundred thirty-eight patients underwent operative laparoscopy for removal of suspicious adnexal masses. The CA 125 level was >35 mIU/ml in 39 of 138 (28%) patients; ultrasonographic findings were abnormal in 127 of 138 (92%); masses were >10 cm in 43 of 138 (32%) of patients. RESULTS: Malignancies were discovered in 14% (19/138) of patients. Eight percent (11/138) of the procedures were converted to laparotomy, six because of inability to dissect the mass laparoscopically and five for staging or debulking of carcinoma. Operative times ranged from 25 to 210 minutes, with a mean of 86. Three major complications were encountered-an enterotomy and a lacerated vena cava, both of which were repaired laparoscopically, and a small bowel herniation through a lateral port site that required reoperation. Hospital stays ranged from 0 to 11 days, with a mean of 1.5. In two patients with "apparent" stage I adnexal carcinomas recurrence was diagnosed 6 and 38 months after surgery. CONCLUSIONS: Laparoscopic management of suspicious adnexal masses is technically feasible, with a low rate of morbidity and a short hospital stay. Adnexal carcinomas can be identified and managed appropriately with staging and complete resection as indicated. (Am J Obstet Gynecol 1996;175:1451-9.)     

Abrogation of head and neck squamous cell carcinoma growth by epidermal growth factor receptor ligand fused to pseudomonas exotoxin transforming growth factor alpha-PE38.

PURPOSE: This study was undertaken to determine whether low intratumoral doses of the epidermal growth factor receptor ligand-transforming growth factor alpha (TGF-alpha) fused to Pseudomonas exotoxin (TGF-alpha-PE38)-abrogated head and neck squamous cell carcinoma (HNSCC) tumor growth in vitro and in vivo. EXPERIMENTAL DESIGN: In vitro cytotoxicity assays were carried out to determine the sensitivity of HNSCC cells to TGF-alpha-PE38. TGF-alpha-PE38-treated HNSCC cells were examined by immunoblotting for cleaved poly(ADP-ribose) polymerase to evaluate apoptosis. Nude mice bearing established HNSCC xenografts were treated with several doses of TGF-alpha-PE38 to evaluate the antitumor efficacy in vivo. Tumor sections were stained with terminal deoxynucleotidyl transferase-mediated nick end labeling for apoptosis. To determine the effect of oral administration of TGF-alpha-PE38, gavage injections of TGF-alpha-PE38 were administered, and the esophagus and surrounding soft tissue were then stained for apoptotic cells. RESULTS: HNSCC cell lines examined were sensitive to low doses of TGF-alpha-PE38 (EC(50) in the range of 1.6 to 10 ng/mL). HNSCC cells treated with TGF-alpha-PE38 undergo apoptosis. Antitumor effects were observed using 0.1 and 0.03 microg of TGF-alpha-PE38 administered intratumorally. At these doses, the treatment was well tolerated. Tumors treated with the toxin had a higher number of apoptotic cells compared with the control tumors. No apoptotic cells were observed in the pharyngoesophageal tissues of the mice after gavage administration of the toxin suggesting that the toxin could be orally administered without toxicity. CONCLUSIONS: These results indicate that topical or intratumoral administration of low doses of TGF-alpha-PE38 may demonstrate antitumor effects in HNSCC without associated systemic toxicity.     

A phase I trial of the dual farnesyltransferase and geranylgeranyltransferase inhibitor L-778,123 and radiotherapy for locally advanced pancreatic cancer.

PURPOSE: Preclinical and clinical studies have demonstrated that inhibition of prenylation can radiosensitize cell lines with activation of Ras and produce clinical response in patients with cancer. The aim of this study was to determine the maximally tolerated dose of the dual farnesyltransferase and geranylgeranyltransferase I inhibitor L-778,123 in combination with radiotherapy for patients with locally advanced pancreatic cancer. EXPERIMENTAL DESIGN: L-778,123 was given by continuous intravenous infusion with concomitant radiotherapy to 59.4 Gy in standard fractions. Two L-778,123 dose levels were tested: 280 mg/m2/day over weeks 1, 2, 4, and 5 for dose level 1; and 560 mg/m2/day over weeks 1, 2, 4, 5, and 7 for dose level 2. RESULTS: There were no dose-limiting toxicities observed in the eight patients treated on dose level 1. Two of the four patients on dose level 2 experienced dose-limiting toxicities consisting of grade 3 diarrhea in one case and grade 3 gastrointestinal hemorrhage associated with grade 3 thrombocytopenia and neutropenia in the other case. Other common toxicities were mild neutropenia, dehydration, hyperglycemia, and nausea/vomiting. One patient on dose level 1 showed a partial response of 6 months in duration. Both reversible inhibition of HDJ2 farnesylation and radiosensitization of a study patient-derived cell line were demonstrated in the presence of L-778,123. K-RAS mutations were found in three of the four patients evaluated. CONCLUSIONS: The combination of L-778,123 and radiotherapy at dose level 1 showed acceptable toxicity in patients with locally advanced pancreatic cancer. Radiosensitization of a patient-derived pancreatic cancer cell line was observed.     

SARS-CoV-2 Variants in Paraguay: Detection and Surveillance with an Economical and Scalable Molecular Protocol

SARS-CoV-2 variant detection relies on resource-intensive whole-genome sequencing methods. We sought to develop a scalable protocol for variant detection and surveillance in Paraguay, pairing rRT-PCR for spike mutations with Nanopore sequencing. A total of 201 acute-phase nasopharyngeal samples were included. Samples were positive for the SARS-CoV-2 N2 target and tested with the Spike SNP assay to detect mutations associated with the following variants: alpha (501Y), beta/gamma (417variant/484K/501Y), delta (452R/478K), and lambda (452Q/490S). Spike SNP calls were confirmed using amplicon (Sanger) sequencing and whole-genome (Nanopore) sequencing on a subset of samples with confirmed variant lineages. Samples had a mean N2 Ct of 20.8 (SD 5.6); 198/201 samples (98.5%) tested positive in the Spike SNP assay. The most common genotype was 417variant/484K/501Y, detected in 102/198 samples (51.5%), which was consistent with the P.1 lineage (gamma variant) in Paraguay. No mutations (K417 only) were found in 64/198 (32.3%), and K417/484K was identified in 22/198 (11.1%), consistent with P.2 (zeta). Seven samples (3.5%) tested positive for 452R without 478K, and one sample with genotype K417/501Y was confirmed as B.1.1.7 (alpha). The results were confirmed using Sanger sequencing in 181/181 samples, and variant calls were consistent with Nanopore sequencing in 29/29 samples. The Spike SNP assay could improve population-level surveillance for mutations associated with SARS-CoV-2 variants and inform the judicious use of sequencing resources.     

Bioethics as care work

German philosopher Martin Heidegger argued that humans are defined by care. The term he used, “Sorge,” picks out a wide range of caring relations, including sorrow, worry, the making of arrangements, and even fending for another. Since coming to The Hastings Center, I've been struck by the genuine care definitive of its scholars’ relationship to their work. Care about newborns, the elderly, and nonhuman animals. Care about doctors, nurses, and health care institutions. Care expressed in the panoply of ways biomedical knowledge and practices inform our havings, doings, and beings in the world. Perhaps in its better moments, bioethics just is care work. But care work is hard and messy.