Subsequent Renal Cancer Among Childhood Cancer Survivors: Analysis of Surveillance, Epidemiology, and End Results.

Renal cancer, although still rare among individuals under 45 years of age, is on the rise in the general population. The risk and timing of subsequent renal cancer in survivors of childhood cancer is not well established. Using the SEER registry, we reported the incidence of subsequent malignant renal neoplasms after treatment for primary malignancy diagnosed under 20 years of age. We evaluated clinical characteristics, standardized incidence ratio (SIR), and Kaplan-Meier survival estimates. Fifty-three survivors developed subsequent renal cancer (54 total cases). Of these, 54.7% were female, 88.7% were white, and 13.2% were Hispanic. Mean ages at primary malignancy and subsequent renal cancer were 10.1 and 31.1 years, respectively. Forty-seven cases were second cancers, 6 were third, and 1 was fourth. For survivors of childhood cancer, the overall SIR for renal cancer was 4.52 (95% CI: 3.39-5.89). The 5-year overall survival rate after development of subsequent renal cancer was 73% (95% CI: 58%-83%). Renal cancer occurs 4.5 times more frequently in childhood cancer survivors than in the general population, necessitating long-term care considerations.

The expression of 'policy' in palliative care: A critical review.

The importance of 'policy' within palliative care has steadily increased over the past 25 years. Whilst this has been welcomed within the palliative care field and seen as a route to greater recognition, we focus here on a more critical perspective that challenge the effectiveness of a 'policy turn' in palliative care. Applying Bacchi's "What's the Problem Represented to Be?" (WPR) framework to data from a systematic search, we address the research question, "in what ways has 'policy' been articulated in palliative care literature?". The paper describes the construction of 'the problem' context and reflects critically on the robustness and pragmatic utility of such representations. In particular, we identify five elements as prominent and problematic: (1) a lack of empirical evidence that connects policy to practice; (2) the dominance of 'Global North' approaches; (3) the use of a policy narrative based on 'catastrophe' in justifying the need for palliative care; (4) the use of idealistic and aspirational 'calls to action'; and (5) a disengaged and antagonistic orientation to existing health systems. We conclude by suggesting that the efficacy of palliative care policy could be enhanced via greater emphases on 'Global South' perspectives, 'assets-based' approaches and attention to pragmatic implementation.

Clinical validation and implementation of droplet digital PCR for the detection of BRAF mutations from cell-free DNA.

Droplet digital PCR (ddPCR) has been demonstrated in many research studies to be a sensitive method in the analysis of circulating tumour DNA (ctDNA) for identifying mutations and tracking disease. The transition of ddPCR into the diagnostic setting requires a number of critical steps including the assessment of accuracy and precision and ultimately implementation into clinical use. Here we present the clinical validation of ddPCR for the detection of BRAF mutations (V600E and V600K) from plasma. We describe the performance characteristics assessed including the limit of blank, limit of detection, ruggedness, accuracy, precision and the effect of the matrix. Overall, each assay could achieve a limit of detection of 0.5% variant allele fraction and was highly accurate, with 100% concordance of results obtained from routine diagnostic testing of formalin fixed tumour samples or reference controls (n=36 for BRAF V600E and n=30 for BRAF V600K). Inter-laboratory reproducibility across 12 plasma samples for each assay was also assessed and results were 100% concordant. Overall, we report the successful validation and translation of a ddPCR assay into clinical routine practice.

Influence of opioid limiting legislation on patient narcotic usage and provider burden in orthopedic trauma.

OBJECTIVE: Opiate consumption in the United States has reached alarming levels. As a result, the state of Florida enacted House Bill 21 (HB21) in July 2018. Following HB21, we hypothesized total opioids prescribed would decrease, with a resulting increase in phone calls, unscheduled visits for pain control, and refills dispensed. DESIGN: Retrospective cohort study comparing opiate usage 6 months before and after HB21 enactment. SETTING: Single Level I academic trauma center. PARTICIPANTS: Patients with isolated lower extremity fractures who were treated with a single surgery. INTERVENTION: Opioid prescription limitations according to Florida's HB21. MAIN OUTCOME MEASURES: We compared morphine milligram equivalents (MMEs) dispensed at discharge, length of stay (LOS), readmissions, emergency room (ER) visits, calls for pain control, refills, and total MMEs dispensed for 3 months postoperatively. RESULTS: A total of 116 patients met inclusion criteria. Our results demonstrated a decrease in the median MMEs provided at discharge (288 vs. 184, p 0.005) and total MMEs dispensed (375 vs. 225, p 0.0003). There was no significant difference in LOS (2 vs. 2.5 days, p 0.979), unscheduled clinic visits for pain (two per group), ER visits for pain (eight per group), or phone calls for pain (13 vs. 9, p 0.344). There were no readmissions for pain control pre-HB21 and one post-HB21. The percentage of patients obtaining >1 refill decreased from 22.4 to 1.7 percent (p 0.002). CONCLUSIONS: Legislation restricting opioid pain medications may be effective in decreasing opiate use in orthopedic trauma patients while decreasing provider burden.

How the Reputation of Orthopaedic Residency Programs Is Associated with Orthopaedic Fellowship Match Results.

BACKGROUND: The primary goal of the present study was to determine if applicants from higher-ranking U.S. orthopaedic surgery residency programs match at a more desired position on their fellowship match-rank list compared with those applicants from lower-ranked residency programs. METHODS: San Francisco Match provided results regarding applicant data and match results from 2014 to 2018 for all orthopaedic subspecialties except the hand and the shoulder and elbow. Unmatched applicants and international medical graduates were excluded. Residency programs were divided into 5 tiers (with tier 1 being the highest-ranked residency programs and tier 5 being the lowest-ranked programs) on the basis of 2018 Doximity rankings of orthopaedic residency programs. Statistical analysis consisted of descriptive statistics, chi-square tests, and analysis of variance. RESULTS: Two thousand eight hundred and eleven applicants met inclusion criteria. Applicants from residency programs in tiers 1 and 2 applied to significantly fewer programs than those from tiers 3, 4, or 5 (p < 0.0001). Applicants from each tier were significantly more likely to attain interviews than applicants from all tiers below them (p < 0.01). Applicants from tier-1 residency programs matched at a significantly higher position on their rank list (p < 0.001) and were more desirably ranked by fellowship programs (p = 0.003) compared with all other tiers. CONCLUSIONS: Applicants from the highest-ranking residency programs apply to fewer programs, interview at a greater percentage of these programs, and are more likely to match to 1 of their top-ranking programs than applicants from lower-ranking programs. However, the association of the applicant match position with the program ranking appears to be most pronounced when it comes to fellowships selecting which applicants to interview. These findings may help future applicants when determining which programs to apply to during the match.

Controlled substance prescribing and education in orthopedic residencies: A program director survey.

BACKGROUND: Opioid misuse is currently plaguing the US. Efforts to reduce this include opioid prescribing education (OPE). Orthopaedic residents often prescribe opioids but, their education is unknown. METHODS: A survey was sent to orthoapedic residency program directors (PDs) regarding their program's controlled substance (CS) policies and knowledge of local CS regulations. RESULTS: There were 60 (36.8%) completed surveys. 54 (90.0%) programs allow resident outpatient opioid prescribing. Nine (16.7%) programs require individual DEA registration and 7 (13.0%) were unsure about DEA registrations. State laws regarding PDMP utilization and OPE for fully licensed physicians were correctly answered by 52 (86.7%) and 43 (71.6%), respectively. 27 (45.0%) programs had a mandatory OPE. Six (10.0%) PDs were unsure about a mandatory OPE. 16 (48.5%) programs that did not confirm an OPE were considering adding one. CONCLUSIONS: The majority of programs permit residents outpatient opioid prescribing; less than half provide mandatory OPE. Several PDs were unaware local CS prescribing regulations and education. This study demonstrates opportunities to improve OPE among orthopaedic residencies and PDs' knowledge regarding CS regulations.

Profound MEK inhibitor response in a cutaneous melanoma harboring a GOLGA4-RAF1 fusion.

BRAF and CRAF are critical components of the MAPK signaling pathway which is activated in many cancer types. In approximately 1% of melanomas, BRAF or CRAF are activated through structural arrangements. We describe here a metastatic melanoma with a GOLGA4-RAF1 fusion and pathogenic variants in CTNNB1 and CDKN2A. Anti-CTLA4/anti-PD1 combination immunotherapy failed to control tumor progression. In the absence of other actionable variants the patient was administered MEK inhibitor therapy on the basis of its potential action against RAF1 fusions. This resulted in a profound and clinically significant response. We demonstrated that GOLGA4-RAF1 expression was associated with ERK activation, elevated expression of the RAS/RAF downstream co-effector ETV5, and a high Ki67 index. These findings provide a rationale for the dramatic response to targeted therapy. This study shows that thorough molecular characterization of treatment-resistant cancers can identify therapeutic targets and personalize management, leading to improved patient outcomes.

PathOS: a decision support system for reporting high throughput sequencing of cancers in clinical diagnostic laboratories.

BACKGROUND: The increasing affordability of DNA sequencing has allowed it to be widely deployed in pathology laboratories. However, this has exposed many issues with the analysis and reporting of variants for clinical diagnostic use. Implementing a high-throughput sequencing (NGS) clinical reporting system requires a diverse combination of capabilities, statistical methods to identify variants, global variant databases, a validated bioinformatics pipeline, an auditable laboratory workflow, reproducible clinical assays and quality control monitoring throughout. These capabilities must be packaged in software that integrates the disparate components into a useable system. RESULTS: To meet these needs, we developed a web-based application, PathOS, which takes variant data from a patient sample through to a clinical report. PathOS has been used operationally in the Peter MacCallum Cancer Centre for two years for the analysis, curation and reporting of genetic tests for cancer patients, as well as the curation of large-scale research studies. PathOS has also been deployed in cloud environments allowing multiple institutions to use separate, secure and customisable instances of the system. Increasingly, the bottleneck of variant curation is limiting the adoption of clinical sequencing for molecular diagnostics. PathOS is focused on providing clinical variant curators and pathology laboratories with a decision support system needed for personalised medicine. While the genesis of PathOS has been within cancer molecular diagnostics, the system is applicable to NGS clinical reporting generally. CONCLUSIONS: The widespread availability of genomic sequencers has highlighted the limited availability of software to support clinical decision-making in molecular pathology. PathOS is a system that has been developed and refined in a hospital laboratory context to meet the needs of clinical diagnostics. The software is available as a set of Docker images and source code at https://github.com/PapenfussLab/PathOS .

APC Injuries With Symphyseal Fixation: What Affects Outcome?

OBJECTIVE: To evaluate the influence of the symphyseal position at union, implant failure, and the type of posterior ring injury on validated outcome measures. DESIGN: Retrospective review with prospectively collected validated outcome data. SETTING: Two academic level 1 trauma centers. PATIENTS/PARTICIPANTS: We evaluated 54 patients with operatively treated anterior-posterior compression (APC) type 2 and 3 injuries. INTERVENTION: Thirty-five APC type 2 and 19 APC type 3 injuries were reviewed at a minimum of 2 years after surgery. Average follow-up was 7 years. MAIN OUTCOME MEASURES: Patients were evaluated with validated EuroQol five dimensions (EQ5D), EuroQol health index, Visual Analog Score (VAS) pain, Majeed pelvic scores, and change in work status. The final anterior-posterior (AP) radiograph available was reviewed for implant failure and displacement. Revision surgery was documented based on implant status and displacement at final follow-up. RESULTS: There were trends toward better outcomes for APC type 2 for EQ5D and VAS pain. Patients with injury severity score (ISS) >16 had worse reported health, Majeed scores, and VAS pain. Nineteen patients had failure of fixation. There were no differences in any outcome measure; trends toward better Majeed score were found for patients with intact fixation. Displacements >15 mm anteriorly at final follow-up negatively affect outcomes with significantly worse EQ5D, reported health, and Majeed score. Two patients required revision surgery. There were no differences in final outcomes. CONCLUSIONS: No significant differences were found for APC type 2 versus type 3 injuries. Higher injury severity score resulted in worse outcomes and more pain. Outcomes were not effected by implant failure; however, major loss of reduction (>15 mm) anteriorly did negatively impact outcomes. Patients with failure who were revised to union did not have worse outcomes. LEVEL OF EVIDENCE: Prognostic level IV. See Instructions for Authors for a complete description of levels of evidence.

Molecular methods for somatic mutation testing in lung adenocarcinoma: EGFR and beyond.

Somatic mutational profiling in cancer has revolutionized the practice of clinical oncology. The discovery of driver mutations in non-small cell lung cancer (NSCLC) is an example of this. Molecular testing of lung adenocarcinoma is now considered standard of care and part of the diagnostic algorithm. This article provides an overview of the workflow of molecular testing in a clinical diagnostic laboratory discussing in particular novel assays that are currently in use for somatic mutation detection in NSCLC focussing on epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK), ROS1 and RET rearrangements.

Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing.

BACKGROUND: Clinical specimens undergoing diagnostic molecular pathology testing are fixed in formalin due to the necessity for detailed morphological assessment. However, formalin fixation can cause major issues with molecular testing, as it causes DNA damage such as fragmentation and non-reproducible sequencing artefacts after PCR amplification. In the context of massively parallel sequencing (MPS), distinguishing true low frequency variants from sequencing artefacts remains challenging. The prevalence of formalin-induced DNA damage and its impact on molecular testing and clinical genomics remains poorly understood. METHODS: The Cancer 2015 study is a population-based cancer cohort used to assess the feasibility of mutational screening using MPS in cancer patients from Victoria, Australia. While blocks were formalin-fixed and paraffin-embedded in different anatomical pathology laboratories, they were centrally extracted for DNA utilising the same protocol, and run through the same MPS platform (Illumina TruSeq Amplicon Cancer Panel). The sequencing artefacts in the 1-10% and the 10-25% allele frequency ranges were assessed in 488 formalin-fixed tumours from the pilot phase of the Cancer 2015 cohort. All blocks were less than 2.5 years of age (mean 93 days). RESULTS: Consistent with the signature of DNA damage due to formalin fixation, many formalin-fixed samples displayed disproportionate levels of C>T/G>A changes in the 1-10% allele frequency range. Artefacts were less apparent in the 10-25% allele frequency range. Significantly, changes were inversely correlated with coverage indicating high levels of sequencing artefacts were associated with samples with low amounts of available amplifiable template due to fragmentation. The degree of fragmentation and sequencing artefacts differed between blocks sourced from different anatomical pathology laboratories. In a limited validation of potentially actionable low frequency mutations, a NRAS G12D mutation in a melanoma was shown to be a false positive. CONCLUSIONS: These findings indicate that DNA damage following formalin fixation remains a major challenge in laboratories working with MPS. Methodologies that assess, minimise or remove formalin-induced DNA damaged templates as part of MPS protocols will aid in the interpretation of genomic results leading to better patient outcomes.

A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma.

Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3-4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics.

Factors associated with failure to successfully complete a procedure during emergency department sedation.

OBJECTIVE: To determine factors associated with failure to successfully complete a procedure during sedation in the ED. METHODS: Eleven Australian EDs enrolled consecutive adult and paediatric patients between January 2006 and December 2008. Patients were included if a sedative drug was administered for an ED procedure and the success or failure of the procedure was recorded. RESULTS: Data were available for 2567 patients. Of these, 1548 (60.3%, 95% CI 58.4-62.2) were male and 456 (17.8%, 95% CI 16.3-19.3) were age <16 years. The most common procedures performed were reduction of major joints and laceration repair. A total of 149 procedures (5.8%, 95% CI 5.0-6.8) failed. There were significant differences in failure rates between the types of procedure undertaken, with reduction of hips, digits and mandibles associated with the highest failure rates (P < 0.001). In adults, body weight >100 kg was also associated with increased risk of procedural failure (odds ratio 2.3, 95% CI 1.3-4.1). Ketamine used as a single agent had the lowest procedural failure rate (2.5%, 95% CI 1.1-5.4) whereas propofol had the highest (5.9%, 95% CI 4.6-7.6). However, these two drugs were generally used in different age groups and for different procedures. CONCLUSIONS: Procedures performed under sedation in the ED have a low failure rate. However, increased body weight and specific procedures, such as hip reduction, are associated with significantly higher failure rates. Special consideration should be given to these patient groups before undertaking sedation in the ED.

Treatment of recalcitrant, multiply operated tibial nonunions with the RIA graft and rh-BMP2 using intramedullary nails.

Nonunions of the tibia continue to present some of the most difficult challenges in orthopaedic fracture care. Whether the consequence of the initial presenting injury, co-morbidity or subsequent attempts at fixation, the biological environment is often compromised. Compounding this problem is a lack of consensus on the best approach to addressing nonunited tibia fractures, placing them at risk for multiple, and sometimes ill-informed attempts at nonunion repair. We present nine cases of recalcitrant tibial nonunions which had previously undergone 4 or more attempts at repair treated with a protocol using RIA graft, rh-BMP2 and intramedullary nail fixation.

RD114 envelope proteins provide an effective and versatile approach to pseudotype lentiviral vectors.

Lentiviral vectors derived from the HIV-1 genome offer great promise for gene therapy due to their ability to transduce non-dividing cells and sustain long-term expression of transgenes. The majority of current lentiviral vectors are pseudotyped with the vesicular stomatitis viral envelope (VSV-G). VSV-G equips lentiviral vectors with a broad host cell tropism and increased stability. Increased particle stability enables viral supernatants to be concentrated by high-speed centrifugation to enhance their infectivity. Despite its efficacy, VSV-G is cytotoxic - a feature that prohibits the development of stable cell lines that constitutively express this envelope. Therefore, non-toxic envelope proteins are being investigated. RD114 is an attractive alternative because it also provides increased particle stability and its receptor is widely expressed on hematopoietic stem cells (HSCs). In this study, the packaging efficiency of three envelope proteins, RD114, RDpro and VSV-G, were evaluated with two lentiviral vectors (TRIP GFP and HPV-402). RDpro is an RD114-HIV chimera designed to pseudotype lentiviral vectors more efficiently. In transient systems, VSV-G generated titers of 10(8) and 10(7) viral particles/mL for TRIP GFP and HPV-402. RDpro possessed titers of 10(7) and 10(6), while RD114 titers were one log lower for each vector. Despite having relatively lower titers, RD114 proteins are less toxic; this was demonstrated in the extension of transient transfection reactions from 48 to 96 h. VSV-G transfections are generally limited to 48 h. In regard to gene therapy applications, we show that RDpro supernatants efficiently transduce peripheral blood HSCs. The versatility of RD114 envelopes was again demonstrated by using a 'mixed' expression system; composed of stably expressed RD114 envelope proteins to pseudotype lentiviral vectors generated in trans (titer range 10(3)-10(5)). Our data show that RD114 envelope proteins are effective and versatile constructs that could prove to be essential components of therapeutic lentiviral gene transfer systems.

The Atkinson Morley's Hospital joint neurosurgical-maxillofacial procedures: cranioplasty case series 1985-2003.

AIMS: To present a retrospective case series resulting from the co-operation in cranioplasty procedures between neurosurgeons and maxillofacial surgeons of the Atkinson Morley's and Royal Marsden Hospitals of London, UK for the period 1985-2003. MATERIALS AND METHODS: The cranioplasty case series is part of an integrated analysis of the complete Atkinson Morley's Hospital craniofacial procedure database. Cases included both cosmetic and functional procedures. The latter followed a variety of conditions such as tumour recurrence, craniectomy-associated neurological symptoms, wound infection and infection of previous prosthesis or bone flap. RESULTS: Fourty-eight procedures (27 functional, 21 cosmetic) were performed in 43 patients (mean age: 44.99 years, SD: 18.1 years). Our case notes analysis reviewed symptoms on presentation and duration, previous neurosurgical procedures and previous histopathology, nature and length of operative procedure, imaging studies, post-operative complications and management, and patient follow-up and survival data. CONCLUSION: Analysis of the international literature highlights the paucity and poor quality of evidence on the subject of cranioplasty. The authors hope this work adds to the body of knowledge, despite its retrospective nature.

Genetic heterogeneity of granulocytes for the JAK2 V617F mutation in essential thrombocythaemia: implications for mutation detection in peripheral blood.

AIMS: The molecular pathogenesis of essential thrombocythaemia (ET) is heterogeneous. We aimed to determine the relative sensitivity of four separate molecular assays used to detect the presence of the JAK2 V617F mutation in peripheral blood from patients with essential thrombocythaemia and related myeloproliferative disorders. METHODS: Purified granulocytes from 60 patients were analysed for the presence of the JAK2 V617F mutation by direct sequencing, denaturing high-performance liquid chromatography (DHPLC), allele-specific polymerase chain reaction (PCR) and allele-specific enrichment. Clinical data were collected for all patients and correlated with assay results. RESULTS: Direct sequencing and DHPLC were relatively insensitive assays for mutation detection, together identifying only 53% of the JAK2 V617F positive cases of ET. Allele-specific PCR and allele-specific enrichment were significantly more sensitive assays and were able to identify additional ET patients that were positive for the JAK2 V617F mutation in only a minority of circulating granulocytes. Enrichment for the mutation was demonstrated in blood platelets from two of these patients. CONCLUSIONS: The observed biological difference in circulating granulocyte involvement by the JAK2 V617F clone necessitates a sensitive molecular assay for the diagnostic investigation of thrombocytosis.

A stable murine-based RD114 retroviral packaging line efficiently transduces human hematopoietic cells.

Several barriers exist to high-efficiency transfer of therapeutic genes into human hematopoietic stem cells (HSCs) using complex oncoretroviral vectors. Human clinical trials to date have used Moloney leukemia virus-based amphotropic and gibbon ape leukemia virus-based envelopes in stable retroviral packaging lines. However, retroviruses pseudotyped with these envelopes have low titers due to the inability to concentrate viral supernatants efficiently by centrifugation without damaging the virus and low transduction efficiencies because of low-level expression of viral target receptors on human HSC. The RD114 envelope from the feline endogenous virus has been shown to transduce human CD34+ cells using transient packaging systems and to be concentrated to high titers by centrifugation. Stable packaging systems have potential advantages over transient systems because greater and more reproducible viral productions can be attained. We have, therefore, constructed and tested a stable RD114-expressing packaging line capable of high-level transduction of human CD34+ cells. Viral particles from this cell line were concentrated up to 100-fold (up to 10(7) viral particles/ml) by ultracentrifugation. Human hematopoietic progenitors from cord blood and sickle cell CD34+ cells were efficiently transduced with a Neo(R)-containing vector after a single exposure to concentrated RD114-pseudotyped virus produced from this cell line. Up to 78% of progenitors from transduced cord blood CD34+ cells and 51% of progenitors from sickle cell CD34+ cells expressed the NeoR gene. We also show transfer of a human beta-globin gene into progenitor cells from CD34+ cells from sickle cell patients with this new RD114 stable packaging system. The results indicate that this packaging line may eventually be useful in human clinical trials of globin gene therapy.

In vivo ligation of CD3 on neonatal scid thymocytes blocks gamma-irradiation-induced TCRbeta rearrangements and thymic lymphomagenesis.

Several studies have shown that the developmental arrest of severe combined immune deficiency (scid) thymocytes during the CD4(-)CD8(-) double negative (DN) to CD4(+)CD8(+) double positive (DP) transition can be overcome by a sub-lethal dose of ionizing radiation (IR). Concurrent with this developmental progression, IR also induces variable (diversity) joining (V(D)J) recombination at T cell receptor (TCR), delta, beta, and gamma, but not alpha loci. In addition, all irradiated scid mice succumb to thymic lymphoma. In this study, we demonstrate that scid neonates treated with anti-CD3 epsilon antibody become more resistant to the development of thymoma upon exposure to IR. It is known that the anti-CD3 epsilon antibody treatment induces T cell progression to the DP stage bypassing TCRbeta rearrangement. We show here that the resistance to tumor development is correlated with a reduction of TCRbeta rearrangements that are induced by IR. However, TCRgamma rearrangements were not altered by the antibody treatment. The particular effect of anti-CD3 epsilon antibody on TCRbeta rearrangements is likely attributed to a decline of the double negative thymocyte subset (DN3), in which TCRbeta rearrangements predominantly occur. These results suggest that the developmental stage of scid thymocytes can influence the effect of IR on TCR rearrangements as well as lymphomagenesis.

Non-classical helix-stabilizing interactions: C-H...O H-bonding between Phe and Glu side chains in alpha-helical peptides.

The classical picture of H-bonds has evolved considerably. In contrast to earlier expectations, C-H...O H-bonds are now known to be prevalent in both small organic and large biological systems. However, there are few reports on the energetic contribution of C-H...O H-bonds in protein or polypeptide systems and we do not know whether such interactions are stabilizing. Here we investigate C-H...O H-bonding interactions between Phe and Glu side chains by determining their effects on the helicity of model alpha-helical peptides using a combination of CD and NMR spectroscopy. The results suggest that Glu/Phe C-H...O H-bonding interactions stabilize helical structure, but only in the orientation Glu --> Phe (N --> C). Each Glu --> Phe (N --> C) interaction can contribute approximately -0.5 kcal mol(-1) to the stability of helical peptide. In the reverse orientation, Phe --> Glu (N --> C) appears to contribute negligibly. pH titrations provide further evidence for the existence of C-H...O H-bonds. The C-H...O H-bonding interactions in these peptides are insensitive to the screening effect of added neutral salt. Our results provide quantitative energetic information on C-H...O H-bonds that should be useful for empirical force-field calibration.