Using a digital platform to establish odontometric variation based on race, gender and Angle classification.

Orthodontists often encounter significant clinical challenges in the finishing stages of treatment due to a disproportion in interarch tooth size relationships. Despite the increasing presence of digital technology and concomitant focus on customized treatment approaches, there is a gap in the knowledge of how generating tooth size data using digital versus traditional methods may impact our treatment regime. OBJECTIVE: This study aimed to compare the prevalence of tooth size discrepancies using digital models and a digitally based cast analysis in our cohort based on (i) Angle's Classification; (ii) gender and (iii) race. MATERIALS AND METHODS: The mesiodistal widths of teeth in 101 digital models were assessed using computerized odontometric software. A Chi-square test was used to determine the prevalence of tooth size disproportions among the study groups. The differences between all three groups of the cohort were analysed using a three-way analysis of variance (ANOVA). RESULTS: An overall Bolton tooth size discrepancy (TSD) prevalence of 36.6% was observed in our study cohort; 26.7% had an anterior Bolton TSD. No differences existed in the prevalence of tooth size discrepancies between male and female subjects as well as between the different malocclusion groups (P > .05). Caucasian subjects had a statistically significant smaller prevalence of TSD compared to Black and Hispanic patients (P < .05). CONCLUSION: The prevalence results in this study illuminate how relatively common TSD is and underscores the importance of proper diagnosis. Our findings also suggest that racial background may be an influential factor in the presence of TSD.

Preface to the 9th Biennial COAST Conference: Harnessing Technology and Biomedicine for Personalized Orthodontics.

OBJECTIVE: The Consortium on Orthodontic Advances in Science and Technology (COAST) convened for its 9th biennial conference titled 'Harnessing Technology and Biomedicine for Personalized Orthodontics' to explore cutting-edge craniofacial research towards building the foundations for precision care in orthodontics. SETTING AND SAMPLE POPULATION: Seventy-five faculty, scholars, private practitioners, industry, residents and students met at the UCLA Arrowhead Lodge on 6-9 November 2022 for networking, scientific presentations and facilitated discussions. Thirty-three speakers provided state-of-the-art, evidence-based scientific and perspective updates in craniofacial and orthodontic-related fields. The overall format included an Education Innovation Award Faculty Development Career Enrichment (FaCE) workshop focused on faculty career development, three lunch and learns, keynote or short talks and poster presentations. MATERIAL AND METHODS: The 2022 COAST Conference was organized thematically to include (a) genes, cells and environment in craniofacial development and abnormalities; (b) precision modulation of tooth movement, retention and facial growth; (c) applications of artificial intelligence in craniofacial health; (d) precision approaches to Sleep Medicine, OSA and TMJ therapies; and (e) precision technologies and appliances. RESULTS: The collective advances in orthodontics and science represented in the manuscripts of this issue fulfil our goal of laying solid foundations for personalized orthodontics. Participants elevated the need for stronger industry-academic research partnerships to leverage knowledge gained from large datasets with treatment approaches and outcomes; systematizing the potential of big data including through multi-omics and artificial intelligence approaches; refining the genotype: phenotype correlation to create biotechnology that will rescue inherited dental and craniofacial defects; evolving studies of tooth movement, sleep apnoea and TMD treatment to accurately measure dysfunction and treatment successes; and maximizing the integration of newer orthodontic devices and digital workflows. CONCLUSIONS: Technological advances combined with those in biomedicine and machine learning are rapidly changing the delivery of health care including that in orthodontics. These advances promise to lead to enhanced customization, efficiencies and outcomes of patient care in routine orthodontic problems and in severe craniofacial problems, OSA and TMD.

Facilitators and barriers to attending postpartum screening in women with a recent pregnancy complicated by gestational diabetes mellitus: a qualitative study.

OBJECTIVE: Pregnant women with gestational diabetes mellitus (GDM) are 50% more likely to develop type II diabetes (T2D) within 6 months to 2 years after giving birth. Therefore, international guidelines recommend it is best practice for women diagnosed with GDM to attend screening for T2D 6-12 weeks postpartum and every 1-3 years thereafter for life. However, uptake of postpartum screening is suboptimal. This study will explore the facilitators of and barriers to attending postpartum screening for T2D that women experience. STUDY DESIGN: This was a prospective qualitative cohort study using thematic analysis. METHODS: A total of 27 in-depth, semistructured interviews were conducted over the telephone with women who had recent GDM. Interviews were recorded and transcribed, and data were analysed using thematic analysis. RESULTS: Facilitators of and barriers to attending postpartum screening were identified at three different levels: personal, intervention, and healthcare systems level. The most common facilitators identified were concern for their own health and having the importance of screening explained to them by a health professional. The most common barriers identified were confusion over the test and COVID-19. CONCLUSION: This study identified several facilitators of and barriers to attending postpartum screening. These findings will help to inform research and interventions for improving rates of attendance at postpartum screening to reduce the subsequent risk of developing T2D.

A new cyte in orthodontics: Osteocytes in tooth movement.

Orthodontic tooth movement (OTM) relies on the orchestration of clinical and biologic events that include the application of clinical force followed by a cascade of cellular and molecular responses. Our understanding about OTM today has evolved from, and is largely based on historic studies. However, the advances in bone biology and clinical orthodontics today continue to pave the pathway towards an improved knowledge base, and state of the art therapeutics in OTM. Osteoblasts and osteoclasts have been the primary cells analyzed in OTM. However, the role of osteocytes, a cell previously thought to be static, should be considered in light of new findings in molecular biological research. Osteocytes are now known to be significant in controlling responses to mechanical forces and therefore may be central to both OTM and normal tooth eruption. In this review, we explore the biology of OTM by focusing specifically on the potential role of osteocytes. Evidence from recent studies reveal that osteocytes have a role in controlling the response to mechanical forces and OTM. We therefore propose that these findings and further research endeavours may shape the future of clinical applications-specifically enhanced outcomes in OTM.

In silico and functional evaluation of PTH1R mutations found in patients with primary failure of eruption (PFE).

OBJECTIVES: The genetic basis of PFE (OMIM ID: 125350) was interrogated using molecular functional studies. PFE is a disorder that results in a poor prognosis in the eruption of teeth and by extension, in treatment with a continuous archwire. We tested the hypothesis that PTH1R mutations result in loss of function due to altered protein structure to determine (i) the fate of a functional PTH1R mutation and (ii) the resulting PTH1R protein structure of each functional mutation. METHODS: We used immunofluorescence assay of COS7 cells that were transfected with either the WT or 1092delG PTH1R mutation sequence to compare the fate of the expressed protein. We also performed in silico analysis of the WT PTH1R and four different functional PTH1R mutations RESULTS: Functional studies (IFA) showed a variation in expression between the WT and mutant PTH1R. Further, in silico analysis showed structural differences between WT and mutant PTH1R proteins, particularly in the regions of the 3rd intracellular loop and the 6th transmembrane domain required for efficient PTH1R function. CONCLUSION: PTH1R mutations identified in PFE likely result from diminished function due to truncation of the protein, lack of efficient G-protein interactions and putatively attenuated signal transduction. By identifying the mode of protein dysfunction, scientist-clinicians are better prepared to recognize and thereby develop improved methods of treatment, starting at the molecular level.

Preface to COAST 2016 innovators' workshop on personalized and precision orthodontic therapy.

OBJECTIVE: A second focused workshop explored how to transfer novel findings into clinical orthodontic practice. SETTING AND SAMPLE POPULATION: Participants met in West Palm Beach (Florida, USA), on 9-11 September 2016 for the Consortium for Orthodontic Advances in Science and Technology 2016 Innovators' Workshop (COAST). Approximately 65 registered attendees considered and discussed information from 27 to 34 speakers, 8 to 15 poster presenters and four lunch-hour focus group leaders. MATERIAL AND METHODS: The innovators' workshops were organized according to five themed sessions. The aims of the discussion sessions were to identify the following: i) the strength and impact of the evidenced-based discoveries, ii) required steps to enable further development and iii) required steps to translate these new discoveries into orthodontic practice. RESULTS: The role of gene-environment interactions that underlie complex craniofacial traits was the focus of several sessions. It was agreed that diverse approaches are called for, such as (i) large-scale collaborative efforts for future genetic studies of complex traits; (ii) deep genome sequencing to address the issues of isolated mutations; (iii) quantifying epigenetic-environmental variables in diverse areas myofascial pain, alveolar remodelling and mandibular growth. Common needs identified from the themed sessions were multiscale/multispecies modelling and experimentation using controlled and quantified mechanics and translation of the findings in bone biology between species. Panel discussions led to the consensus that a consortium approach to establish standards for intra-oral scanning and 3D imaging should be initiated. CONCLUSIONS: Current and emerging technologies still require supported research to translate new findings from the laboratory to orthodontic practice.

Personalized and precision orthodontic therapy.

OBJECTIVE: To bring together orthodontic stakeholders from academics, industry, and private practice for a series of thematically focused workshops to explore and develop the transfer of novel approaches into clinical orthodontic practice. SETTING AND SAMPLE POPULATION: Twenty-seven invited speakers, eight poster presenters, and participants of the Consortium for Orthodontic Advances in Science and Technology (COAST) 2014 Innovators' Workshop at the Eaglewood Resort and Spa, Itasca, Illinois, September 11-14, 2014. MATERIAL AND METHODS: Five themed sessions involving between 4-7 presentations followed by panel discussions were organized. The aims of the discussion sessions were to highlight important findings and consider the strength of evidence for these, indicate next steps and needed research or technological developments to move forward, and to weigh the expected benefits from these findings and steps to implement in clinical practice. RESULTS: Among important areas for attention identified were need for multiscale and multispecies modeling and experimentation for interspecies translation of results; large-scale collaborative efforts within the profession to address the need for adequate sample sizes for future genetic studies of complex traits such as malocclusion; a consortium approach to improve new technologies such as intra-oral scanning and 3D imaging by establishing standards; and harnessing the growing body of knowledge about bone biology for application in orthodontics. CONCLUSIONS: With increased awareness of the potential of current and emerging technologies, translation of personalized and precision approaches in the field of orthodontics holds ever-increasing promise.

Characterization of short root anomaly in a Mexican cohort--hereditary idiopathic root malformation.

OBJECTIVE: The purpose of this study was to systematically characterize individuals with short root anomaly (SRA) without any history of orthodontic treatment. The long-term objective of the study was to improve diagnosis and treatment planning and determine risk factors for developing SRA. SETTING AND SAMPLE POPULATION: Twenty-seven patients including two families and 16 unrelated individuals from (9-48 years) reported to orthodontic and/or dental practitioners within the USA. MATERIALS AND METHODS: Digital panoramic and periapical films were analyzed to document pattern and frequency of SRA-affected teeth. Crown-to-root (CR) ratios of the affected teeth were used to characterize the extent of malformation. Pedigree analysis by inspection was completed for one family to determine pattern of inheritance. RESULTS: Twenty-six of the twenty-seven individuals were of Latino descent, and one was of Filipino descent. Hard tissues including enamel, dentin, pulp chambers and canals, and surrounding soft tissues were normal. We found that 25 of 27 individuals had localized SRA and two Latino individuals had generalized SRA. Teeth were affected bilaterally with maxillary central incisors (~63%) and mandibular second premolars most commonly involved (~33%). Affected teeth had a distinct, similar radiographic appearance; in the generalized cases, there was a more severe affection with larger (~twice) CR ratios. Ninety-four percent of affected individuals did not show a significant difference in the CR ratios at different ages. Pedigree analysis suggests an autosomal dominant inheritance pattern in one family. CONCLUSION: This is the first report to show that SRA occurs more frequently in Latino individuals and has a predilection for anterior teeth. The occurrence of SRA in two families further confirms a hereditary component and supports a distinct nosology and nomenclature, hereditary idiopathic root malformation (HIRM) and warrants further investigation.

Challenges associated with electronic and in-person directly observed therapy during a randomized trial.

BACKGROUND: Electronic directly observed therapy (eDOT) has been proposed as an alternative to traditional in-person DOT (ipDOT) for monitoring TB treatment adherence. Information about the comparative performance and implementation of eDOT is limited.METHODS: The frequency of challenges during DOT, challenge type, and effect on medication observation were documented by DOT method during a crossover, noninferiority randomized controlled trial. A logistic mixed-effects model that adjusted for the study design was used to estimate the percentage of successfully observed doses when challenges occurred.RESULTS: A total of 20,097 medication doses were scheduled for observation with either eDOT (15,405/20,097; 76.7%) or ipDOT (4,692/20,097; 23.3%) for 213 study participants. In total, one or more challenges occurred during 17.3% (2,672/15,405) of eDOT sessions and 15.6% (730/4,692) of ipDOT sessions. Among 4,374 documented challenges, 27.3% (n = 1,192) were characterized as technical, 65.9% (n = 2,881) were patient-related, and 6.9% (n = 301) were program-related. Estimated from the logistic model (n = 6,782 doses, 173 participants), the adjusted percentage of doses successfully observed during problematic sessions was 21.7% (95% CI 11.2-37.8) for eDOT and 4.2% (95% CI 1.1-14.7) for ipDOT.CONCLUSION: Compared to ipDOT, challenges were encountered in a slightly higher percentage of eDOT sessions but were more often resolved to enable successful dose observation during problematic sessions.

Bone marrow mesenchymal/fibroblastic stromal cells induce a distinctive EMT-like phenotype in AML cells.

The development of epithelial-to-mesenchymal transition (EMT) like features is emerging as a critical factor involved in the pathogenesis of acute myeloid leukaemia (AML). However, the extracellular signals and the signalling pathways in AML that may regulate EMT remain largely unstudied. We found that the bone marrow (BM) mesenchymal/fibroblastic cell line HS5 induces an EMT-like migratory phenotype in AML cells. AML cells underwent a strong increase of vimentin (VIM) levels that was not mirrored to the same extent by changes of expression of the other EMT core proteins SNAI1 and SNAI2. We validated these particular pattern of co-expression of core-EMT markers in AML cells by performing an in silico analysis using datasets of human tumours. Our data showed that in AML the expression levels of VIM does not completely correlate with the co-expression of core EMT markers observed in epithelial tumours. We also found that vs epithelial tumours, AML cells display a distinct patterns of co-expression of VIM and the actin binding and adhesion regulatory proteins that regulate F-actin dynamics and integrin-mediated adhesions involved in the invasive migration in cells undergoing EMT. We conclude that the BM stroma induces an EMT related pattern of migration in AML cells in a process involving a distinctive regulation of EMT markers and of regulators of cell adhesion and actin dynamics that should be further investigated. Understanding the tumour specific signalling pathways associated with the EMT process may contribute to the development of new tailored therapies for AML as well as in different types of cancers.

Current status of laparoscopic distal pancreatectomy.

Distal pancreatectomy is the therapeutic option of choice for patients with a benign or malignant lesion located in the body and/or tail of the pancreas when surgical intervention is indicated. With recent advances in and wide spread use of imaging studies, lesions of the pancreas are being diagnosed more commonly and it is likely that this will translate into an increased number of patients undergoing surgical resection. The laparoscopic approach to pancreatic resections has not been adopted as rapidly as it has for most other general surgical procedures. This is despite the fact that the current literature appears to validate laparoscopy as an acceptable and safe approach for distal pancreatectomy in patients with benign lesions, and has demonstrated the known benefits inherent to the laparoscopic technique. These benefits include lower intraoperative blood loss, less pain and analgesic requirements, earlier return of bowel function, and shorter recovery and hospital stay. Yet controversy still exists for the role of laparoscopy in the resection of malignant lesions. Recent reports however, have shown that laparoscopic distal pancreatectomy can safely be performed in known malignancies and, most importantly, after a laparoscopic oncological resection, the oncological benchmarks that have been related to survival, (such as negative surgical margins and number of peripancreatic lymph nodes resected), can also be accomplished. We sought to review the current literature on distal pancreatectomy, specifically the indications, laparoscopic approaches, splenectomy and spleen-preserving techniques, intraoperative and short-term outcomes, morbidity, mortality and oncological outcomes.

Standardized delivery room management for neonates with a prenatal diagnosis of congenital heart disease: A model for improving interdisciplinary delivery room care.

Precise characterization of cardiac anatomy and physiology through fetal echocardiography can predict early postnatal clinical course. Some neonates with prenatally defined critical congenital heart disease have anticipated precipitous compromise during perinatal transition for which specialized, diagnosis-specific delivery room care can be arranged to expeditiously stabilize cardiopulmonary hemodynamics. In this article, we describe our institutional approach to the delivery room care of neonates with prenatally diagnosed congenital heart disease, emphasizing our diagnosis-specific care pathways for newborns with critical disease.

Mechanism and control of tooth eruption: overview and clinical implications.

OBJECTIVES: To review pre- and post-emergent eruption, with particular emphasis on distinguishing isolated molar ankylosis from primary failure of eruption (PFE) and genetic considerations in eruption problems. MATERIAL AND METHODS: Radiographic review of eruption failure patients; animal and human experiments; high precision observations of movements of erupting teeth. RESULTS: In pre-emergent tooth eruption, the controlling element is the rate of resorption of overlying structures. A path is cleared, and then the erupting tooth moves along it. This has clinical importance in recognizing the cause of eruption problems, particularly PFE, in which all teeth distal to the most mesial involved tooth do not erupt or respond to orthodontics. In our study of by far the largest sample of PFE cases yet reported, familial cases of PFE accounted for approximately (1/4) of all cases examined. Candidate genes now are being evaluated. In post-emergent eruption, control seems to be light forces of long duration that oppose eruption, rather than heavy forces of short duration such as those during mastication. Studies of human premolars in their passage from gingival emergence to the occlusal plane show that in this phase eruption occurs only during a few hours in the early evening. The critical hours for eruption parallel the time that growth hormone levels are highest in a growing child. In this stage intermittent force does not affect the rate of eruption, but changes in periodontal blood flow do affect it.

Genetic analysis of familial non-syndromic primary failure of eruption.

OBJECTIVES: While some eruption disorders occur as part of a medical syndrome, primary failure of eruption (PFE) - defined as a localized failure of secondary tooth eruption - exists without systemic involvement. Recent studies support that heredity may play an important role in the pathogenesis of PFE. The objective of our human genetic study is to investigate the genetic contribution to PFE. MATERIALS AND METHODS: Four candidate genes POSTN, RUNX2, AMELX, and AMBN) were investigated because of their relationship to tooth eruption or putative relationship to each other. Families and individuals were ascertained based on the clinical diagnosis of PFE. Pedigrees were constructed and analyzed by inspection to determine the mode of inheritance in four families. The candidate genes were directly sequenced for both unrelated affected individuals and unaffected individuals. A genome wide scan using 500 microsatellite markers followed by linkage analysis was carried out for one family. RESULTS: Pedigree analysis of families suggests an autosomal dominant inheritance pattern with complete penetrance and variable expressivity. Sequence analysis revealed two non-functional polymorphisms in the POSTN gene and no other sequence variations in the remaining candidate genes. Genotyping and linkage analysis of one family yielded a LOD score of 1.51 for markers D13S272; D15S118 and D17S831 on chromosomes 13, 15 and 17 respectively. CONCLUSIONS: While LOD scores were not significant evidence of linkage, extension of current pedigrees and novel SNP chip technology holds great promise for identification of a causative locus for PFE.

Preclinical assessment of the distribution of maraviroc to potential human immunodeficiency virus (HIV) sanctuary sites in the central nervous system (CNS) and gut-associated lymphoid tissue (GALT).

1. Growing knowledge of the pathogenesis of human immunodeficiency virus (HIV)-1 infection has led to the identification of potential virus sanctuary sites within the central nervous system and gut-associated lymphoid tissue. 2. Maraviroc is a novel CCR5 antagonist for the treatment of HIV-1 infection. Disposition studies have been performed within the preclinical testing of maraviroc to determine its distribution to these anatomical sites. 3. Maraviroc, which is a substrate of the efflux transporter P-glycoprotein, shows limited distribution to the central nervous system as evidenced by cerebrospinal fluid concentrations that were 10% of the free plasma concentration following intravenous infusion to rats. Tissue distribution studies also indicated limited distribution of radioactivity into brain tissue of rats. 4. Radioactivity in gut-associated lymphoid tissue lymph nodes exceeded the concentrations in blood and concentrations in the contents of thoracic ducts of the lymphatic system were similar to blood levels following intravenous administration to rats.

Paravertebral blocks reduce the risk of postoperative urinary retention in inguinal hernia repair.

PURPOSE: Inguinal hernia repair and general anesthesia (GA) are known risk factors for urinary retention. Paravertebral blocks (PVBs) have been utilized to facilitate enhanced recovery after surgery. We evaluate the benefit of incorporating PVBs into our anesthetic technique in a large cohort of ambulatory patients undergoing inguinal hernia repair. METHODS: Records of 619 adults scheduled for ambulatory inguinal hernia repair between 2010 and 2015 were reviewed and categorized based on anesthetic and surgical approach [GA and open (GAO), GA and laparoscopic (GAL), PVB and open (PVBO), and GA/PVB and open (GA/PVBO)]. Patients were excluded for missing data, self-catheterization, chronic opioid tolerance, and additional surgical procedures coinciding with hernia repair. Risk factors associated with the primary outcome of urinary retention were examined using logistic regression. RESULTS: PVBO (n = 136) had significantly lower odds than GAO of experiencing urinary retention (odds ratio 0.16; 95% CI 0.05-0.51); overall (P < .01), with 4.4% (n = 6) of the patients in the PVBO group having urinary retention versus 22.6% (n = 7) with GAO. Expressed as intravenous morphine equivalences, the PVBO group had the lowest median opioid use (5 mg), followed by GA, PVB, and open (7.5 mg); GAO 25 mg; and GAL 25 mg. Also, 30% (n = 41) of the PVBO group required no opioid analgesia in the postanesthesia care unit. CONCLUSIONS: PVBs as the primary anesthetic or an adjunct to GA is the preferred anesthetic technique for open inguinal hernia repair as it facilitates enhanced recovery after surgery by decreasing risk of urinary retention, opioid requirements, and length of stay.

Nine Novel PAX9 Mutations and a Distinct Tooth Agenesis Genotype-Phenotype.

Tooth agenesis is one of the most common developmental anomalies affecting function and esthetics. The paired-domain transcription factor, Pax9, is critical for patterning and morphogenesis of tooth and taste buds. Mutations of PAX9 have been identified in patients with tooth agenesis. Despite significant progress in the genetics of tooth agenesis, many gaps in knowledge exist in refining the genotype-phenotype correlation between PAX9 and tooth agenesis. In the present study, we complete genetic and phenotypic characterization of multiplex Chinese families with nonsyndromic (NS) tooth agenesis. Direct sequencing of polymerase chain reaction products revealed 9 novel (c.140G>C, c.167T>A, c.332G>C, c.194C>A, c.271A>T, c.146delC, c.185_189dup, c.256_262dup, and c.592delG) and 2 known heterozygous mutations in the PAX9 gene among 120 probands. Subsequently, pedigrees were extended, and we confirmed that the mutations co-segregated with the tooth agenesis phenotype (with exception of families in which DNA analysis was not available). In 1 family ( n = 6), 2 individuals harbored both the PAX9 c.592delG mutation and a heterozygous missense mutation (c.739C>T) in the MSX1 gene. Clinical characterization of families segregating a PAX9 mutation reveal that all affected individuals were missing the mandibular second molar and their maxillary central incisors are most susceptible to microdontia. A significant reduction of bitter taste perception was documented in individuals harboring PAX9 mutations ( n = 3). Functional studies revealed that PAX9 haploinsufficiency or a loss of function of the PAX9 protein underlies tooth agenesis.

Genomic instability during Myc-induced lymphomagenesis in the bursa of Fabricius.

Retroviral vector-mediated overexpression of c-myc in embryonic bursal precursors induces multi-staged tumorigenesis beginning with preneoplastic-transformed follicles (TF) and progressing to clonal metastatic B-cell lymphomas. Using a 13K chicken cDNA microarray, specifically enriched for chicken immune system expressed sequence tagged (ESTs), we carried out array-based comparative genomic hybridization (array-CGH) and detected significant DNA copy number change at many loci on most or all chromosomes in both early TF and end-stage lymphomas. Formation of long palindromes, through breakage-fusion-bridge cycles, is thought to play an early role in gene amplification. Employing genome-wide analysis of palindrome formation (GAPF), we detected extensive palindrome formation in early TF and end-stage lymphomas. The population of loci showing amplification by array-CGH was enriched for palindromes detected by GAPF providing strong evidence for genetic instability early in Myc-induced tumorigenesis and further support for the role of palindromes in gene amplification. Comparing gene copy number change and RNA expression changes profiled on the same cDNA array, we detected very little consistent contribution of gene copy number change to RNA expression changes. Palindromic loci in TF and tumors, however, were expressed, many at high levels, suggesting an abundance of RNA species with long double-stranded segments generated during tumorigenesis.

Regulation of rat luteinizing hormone subunit messenger ribonucleic acids by gonadal steroid hormones.

Little is known about the hormonal regulation of luteinizing hormone (LH) biosynthesis. We have studied the regulation of LH messenger RNA (mRNA) levels by gonadal-steroid hormones in the rat. In one set of experiments, male and female rats were surgically gonadectomized (GDX) and killed 1, 3, 7, 14, 22, and 31 d postoperatively. In another set of experiments, male and female rats were surgically GDX and were injected subcutaneously with testosterone propionate (500 micrograms/100 g body wt per d) or 17 beta-estradiol 3-benzoate (10 micrograms/100 g body wt per d), respectively, beginning 3 wk postoperatively. Levels of serum LH were determined by radioimmunoassay and levels of LH subunit mRNAs in single pituitary glands were determined by blot hybridization analysis using labeled synthetic oligodeoxyribonucleotide probes that correspond to portions of the coding regions of the rat alpha- and LH beta-subunit mRNAs. 4 wk after gonadectomy, serum LH levels rose nine- and 20-fold, while alpha-subunit mRNA levels rose six- and 10-fold, and LH beta-subunit levels rose seven- and 14-fold, compared with controls in males and females, respectively. In gonadal-steroid hormone-treated male and female GDX rats, serum LH levels fell to 8 and 36% of control values, while alpha-subunit mRNA levels declined to 22 and 19%, and LH beta-subunit mRNA levels declined to 6 and 10% of control values, 48 h after injections were initiated, in males and females, respectively. We conclude that gonadal-steroid hormones negatively regulate the levels of both subunit mRNAs in GDX rats in a pattern that parallels the changes in serum LH values. These data suggest that gonadal-steroid hormone regulation of LH biosynthesis occurs, at least in part, at the level of LH subunit mRNAs due to effects at the transcriptional and/or RNA stability levels.

Biochemical and chemotherapeutic studies on 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine (BW 301U), a novel lipid-soluble inhibitor of dihydrofolate reductase.

The lipophilic diaminopyridopyrimidine BW 301U (2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine) is as active as methotrexate as an inhibitor of dihydrofolate reductase and mammalian cell growth. This compound was selected from among related pyridopyrimidines and other lipid-soluble diaminoheterocyclic compounds as having the most favorable combination of properties as a potent inhibitor of dihydrofolate reductase with minimal effects on histamine metabolism. In contrast to methotrexate, entry of BW 301U into cells is rapid and is not temperature dependent, indicating passage across cell membranes by diffusion. There is no competition between BW 301U and leucovorin (folinic acid) for uptake into Sarcoma 180 cells in culture. When BW 301U is added to culture medium, deoxyuridine incorporation ceases within the first few min, and this inhibition persists when cells are transferred to drug-free medium. Both leucovorin and thymidine are required to protect cells in culture from the cytotoxicity of BW 301U. The effect on thymidine biosynthesis appears to be indirect since BW 301U is inactive as an inhibitor of thymidylate synthetase. Hypoxanthine and thymidine restore growth by only 50% in cultures containing BW 301U, and complete restoration of growth requires the further addition of adenosine and either uridine or cytidine to the medium. In vivo, BW 301U is active against Walker 256, L1210, P388, Sarcoma 180, and Ehrlich ascites tumors.