Genetic variation within the human papillomavirus type 16 genome is associated with oropharyngeal cancer prognosis.

PURPOSE: A significant barrier to adoption of de-escalated treatment protocols for human papillomavirus-driven oropharyngeal cancer (HPV-OPC) is that few predictors of poor prognosis exist. We conducted the first large whole-genome sequencing (WGS) study to characterize the genetic variation of the HPV type 16 (HPV16) genome and to evaluate its association with HPV-OPC patient survival. PATIENTS AND METHODS: A total of 460 OPC tumor specimens from two large United States medical centers (1980-2017) underwent HPV16 whole-genome sequencing. Site-specific variable positions [single nucleotide polymorphisms (SNPs)] across the HPV16 genome were identified. Cox proportional hazards model estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for overall survival by HPV16 SNPs. Harrell C-index and time-dependent positive predictive value (PPV) curves and areas under the PPV curves were used to evaluate the predictive accuracy of HPV16 SNPs for overall survival. RESULTS: A total of 384 OPC tumor specimens (83.48%) passed quality control filters with sufficient depth and coverage of HPV16 genome sequencing to be analyzed. Some 284 HPV16 SNPs with a minor allele frequency ≥1% were identified. Eight HPV16 SNPs were significantly associated with worse survival after false discovery rate correction (individual prevalence: 1.0%-5.5%; combined prevalence: 15.10%); E1 gene position 1053 [HR for overall survival (HRos): 3.75, 95% CI 1.77-7.95; Pfdr = 0.0099]; L2 gene positions 4410 (HRos: 5.32, 95% CI 1.91-14.81; Pfdr = 0.0120), 4539 (HRos: 6.54, 95% CI 2.03-21.08; Pfdr = 0.0117); 5050 (HRos: 6.53, 95% CI 2.34-18.24; Pfdr = 0.0030), and 5254 (HRos: 7.76, 95% CI 2.41-24.98; Pfdr = 0.0030); and L1 gene positions 5962 (HRos: 4.40, 95% CI 1.88-10.31; Pfdr = 0.0110) and 6025 (HRos: 5.71, 95% CI 2.43-13.41; Pfdr = 0.0008) and position 7173 within the upstream regulatory region (HRos: 9.90, 95% CI 3.05-32.12; Pfdr = 0.0007). Median survival time for patients with ≥1 high-risk HPV16 SNPs was 3.96 years compared with 18.67 years for patients without a high-risk SNP; log-rank test P < 0.001. HPV16 SNPs significantly improved the predictive accuracy for overall survival above traditional factors (age, smoking, stage, treatment); increase in C-index was 0.069 (95% CI 0.019-0.119, P < 0.001); increase in area under the PPV curve for predicting 5-year survival was 0.068 (95% CI 0.015-0.111, P = 0.008). CONCLUSIONS: HPV16 genetic variation is associated with HPV-OPC prognosis and can improve prognostic accuracy.

Phenomenological Constraints on the Transport Properties of QCD Matter with Data-Driven Model Averaging.

Using combined data from the Relativistic Heavy Ion and Large Hadron Colliders, we constrain the shear and bulk viscosities of quark-gluon plasma (QGP) at temperatures of ∼150-350 MeV. We use Bayesian inference to translate experimental and theoretical uncertainties into probabilistic constraints for the viscosities. With Bayesian model averaging we propagate an estimate of the model uncertainty generated by the transition from hydrodynamics to hadron transport in the plasma's final evolution stage, providing the most reliable phenomenological constraints to date on the QGP viscosities.

Spin Dependence of η Meson Production in Proton-Proton Collisions Close to Threshold.

Taking advantage of the high acceptance and axial symmetry of the WASA-at-COSY detector, and the high polarization degree of the proton beam of COSY, the reaction p[over →]p→ppη has been measured close to threshold to explore the analyzing power A_{y}. The angular distribution of A_{y} is determined with the precision improved by more than 1 order of magnitude with respect to previous results, allowing a first accurate comparison with theoretical predictions. The determined analyzing power is consistent with zero for an excess energy of Q=15 MeV, signaling s-wave production with no evidence for higher partial waves. At Q=72 MeV the data reveal strong interference of Ps and Pp partial waves and cancellation of (Pp)^{2} and Ss^{*}Sd contributions. These results rule out the presently available theoretical predictions for the production mechanism of the η meson.

Hemodialysis Clinic Social Networks, Sex Differences, and Renal Transplantation.

This study describes patient social networks within a new hemodialysis clinic and models the association between social network participation and kidney transplantation. Survey and observational data collected between August 2012 and February 2015 were used to observe the formation of a social network of 46 hemodialysis patients in a newly opened clinic. Thirty-two (70%) patients formed a social network, discussing health (59%) and transplantation (44%) with other patients. While transplant-eligible women participated in the network less often than men (56% vs. 90%, p = 0.02), women who participated discussed their health more often than men (90% vs. 45.5%, p = 0.02). Patients in the social network completed a median of two steps toward transplantation compared with a median of 0 for socially isolated patients (p = 0.003). Patients also completed more steps if network members were closely connected (ß = 2.23, 95% confidence interval [CI] 0.16-4.29, p = 0.03) and if network members themselves completed more steps (ß = 2.84, 95% CI 0.11-5.57, p = 0.04). The hemodialysis clinic patient social network had a net positive effect on completion of transplant steps, and patients who interacted with each other completed a similar number of steps.

Determination of the η(')-proton scattering length in free space.

Taking advantage of both the high mass resolution of the COSY-11 detector and the high energy resolution of the low-emittance proton beam of the cooler synchrotron COSY, we determine the excitation function for the pp→ppη(') reaction close to threshold. Combining these data with previous results, we extract the scattering length for the η(')-proton potential in free space to be Re(a(pη(')))=0±0.43 fm and Im(a(pη(')))=0.37(-0.16)(+0.40) fm.

Delivery Models and Health Economics of Supportive Care Services in England: A Multicentre Analysis.

AIMS: Improvements in cancer treatment have led to more people living with and beyond cancer. These patients have symptom and support needs unmet by current services. The development of enhanced supportive care (ESC) services may meet the longitudinal care needs of these patients, including at the end of life. This study aimed to determine the impact and health economic benefits of ESC for patients living with treatable but not curable cancer. MATERIALS AND METHODS: A prospective observational evaluation was undertaken over 12 months across eight cancer centres in England. ESC service design and costs were recorded. Data relating to patients' symptom burden were collected using the Integrated Palliative Care Outcome Scale (IPOS). For patients in the last year of life, secondary care use was compared against an NHS England published benchmark. RESULTS: In total, 4594 patients were seen by ESC services, of whom 1061 died during follow-up. Mean IPOS scores improved across all tumour groups. In total, £1,676,044 was spent delivering ESC across the eight centres. Reductions in secondary care usage for the 1061 patients who died saved a total of £8,490,581. CONCLUSIONS: People living with cancer suffer with complex and unmet needs. ESC services appear to be effective at supporting these vulnerable people and significantly reduce the costs of their care.

A Case of HPV-Associated Oropharyngeal Squamous Cell Carcinoma with Block-Like, Partial Loss of p16 Expression.

BACKGROUND: Oropharyngeal squamous cell carcinoma is frequently associated with high-risk HPV infection, which confers a good prognosis. Immunohistochemistry for p16 is used as a surrogate for HPV status, but discrepant results are occasionally seen. Here, we report a case with a unique pattern of partial loss of p16. METHODS: A 63 year old male presented with a base of tongue nonkeratinizing squamous cell carcinoma and a large metastatic neck mass. The primary lesion and multiple regions of the metastatic mass were assessed with p16 immunohistochemistry, RNA in situ hybridization for high-risk HPV, and HPV16 genome sequencing. RESULTS: The primary lesion was p16 negative, and the metastatic neck mass had large, confluent regions that were either strongly p16 positive or entirely p16 negative. All of these regions were positive for high-risk HPV with identical HPV16 genomes. CONCLUSION: This unusual case illustrates a potential diagnostic pitfall, and it raises important questions regarding molecular mechanisms and prognostic implications of p16 staining in oropharyngeal squamous cell carcinoma.

A Short Report Examining the Introduction of Routine Use of Patient-Reported Outcome Measures in a Mixed Oncology Population.

AIMS: People living with treatable but not curable cancer often experience a range of symptoms related to their cancer and its treatment. During the COVID-19 pandemic, face-to-face consultations were reduced and so remote monitoring of these needs was necessary. University Hospitals Sussex implemented the routine use of electronic remote patient-reported outcome measures (PROMs) in a mixed oncology population, focusing on those with treatable but not curable cancers. MATERIALS AND METHODS: Over a 9-month period, patients were invited to register with My Clinical Outcomes (MCO) - a secure online platform for the collection of electronic PROMs. They were prompted by e-mail to complete assessments (EORTC QLQ-C30, EQ-5D-3L and EQ-5D VAS) routinely every 2 weeks. The team monitored patient scores and changes in these prompted clinical interventions. RESULTS: In total, 324 patients completed at least one assessment. The median number of assessments completed by each patient was eight. The most represented tumour groups were secondary breast (28%), prostate (25%) and other (32%). Median scores for the assessments did not deteriorate in a clinically or numerically significant way for patients living with non-curable conditions for the majority of patients monitored. CONCLUSION: Routine collection of electronic remote PROMs is an effective and useful strategy to provide real-time clinical feedback to teams. With integration into existing systems, online platforms (such as MCO) could provide efficient and patient-centred information for those providing care for people with cancer.

Ultrasound imaging of the patellar tendon attachment to the tibia during puberty: a 12-month follow-up in tennis players.

This longitudinal study investigated the ultrasound appearance of the patellar tendon attachment to the tibia throughout puberty in young tennis players with and without Osgood-Schlatter disease (OSD). Twenty-eight competitive players (17 boys), aged 10.6-15.3 years, had bilateral ultrasound imaging of the patellar tendon attachment to the tibia at baseline and 1 year later. On each occasion, anthropometric measurements, pubertal status and injury history were recorded. Ultrasound appearance of the patellar tendon attachment was categorized into three stages: cartilage attachment, insertional cartilage and mature attachment. Stage 1 appearance, a large anechoic region with or without ossicles and irregularity of the apophysis that are classically associated with OSD, was found in eight players, seven of them were pain free. A majority (62%) of the patellar tendons in stage 1 at baseline progressed toward stage 2 or stage 3 1 year later. Likewise the patellar tendon attachment in most athletes with cartilage insertion showed progression to a mature enthesis over 1 year. The imaging appearance that is classically interpreted as OSD was common in asymptomatic knees. This ultrasonographic description of the patellar tendon attachment to the tibia during growth provides a reference for the assessment of bone tendon attachments in adolescents.

Effects of a multi-component exercise program and calcium-vitamin-D3-fortified milk on bone mineral density in older men: a randomised controlled trial.

SUMMARY: We examined the independent and combined effects of a multi-component exercise program and calcium-vitamin-D(3)-fortified milk on bone mineral density (BMD) in older men. Exercise resulted in a 1.8% net gain in femoral neck BMD, but additional calcium-vitamin D(3) did not enhance the response in this group of older well-nourished men. INTRODUCTION: This 12-month randomised controlled trial assessed whether calcium-vitamin-D(3)-fortified milk could enhance the effects of a multi-component exercise program on BMD in older men. METHODS: Men (n = 180) aged 50-79 years were randomised into: (1) exercise + fortified milk; (2) exercise; (3) fortified milk; or (4) controls. Exercise consisted of high intensity progressive resistance training with weight-bearing impact exercise. Men assigned to fortified milk consumed 400 mL/day of low fat milk providing an additional 1,000 mg/day calcium and 800 IU/day vitamin D(3). Femoral neck (FN), total hip, lumbar spine and trochanter BMD and body composition (DXA), muscle strength 25-hydroxyvitamin D and parathyroid hormone (PTH) were assessed. RESULTS: There were no exercise-by-fortified milk interactions at any skeletal site. Exercise resulted in a 1.8% net gain in FN BMD relative to no-exercise (p < 0.001); lean mass (0.6 kg, p < 0.05) and muscle strength (20-52%, p < 0.001) also increased in response to exercise. For lumbar spine BMD, there was a net 1.4-1.5% increase in all treatment groups relative to controls (all p < 0.01). There were no main effects of fortified milk at any skeletal site. CONCLUSION: A multi-component community-based exercise program was effective for increasing FN BMD in older men, but additional calcium-vitamin D(3) did not enhance the osteogenic response.

Mutant Trp repressors with new DNA-binding specificities.

Oligonucleotide-directed mutagenesis of the codons for glutamine-68 (Gln68), lysine-72 (Lys72), isoleucine-79 (Ile79), alanine-80 (Ala80), and threonine-81 (Thr81) of the Escherichia coli trpR (tryptophan aporepressor) gene was used to make mutant repressors with each of 36 different amino acid changes. Mutant repressors were tested for binding to each member of a set of 28 different operators closely related to the consensus trp operator. Of the 36 mutant repressors, 11 bind a subset of the 28 operators; 5 of these have new binding specificities. These new specificities indicate that the hydroxyl group of Thr81 makes a specific contact with one of the four critical base pairs in a trp operator half-site, and the methyl group of Thr81 determines specificity at a second, critical base pair. The Trp repressor does not use the first two amino acids of its "recognition alpha-helix," Ile79 and Ala80, to make sequence-specific DNA contacts, and interacts with its operator in vivo in a way fundamentally different from the way that phage lambda repressor, lambda Cro protein, and coliphage 434 repressor contact their respective binding sites.

Zinc mediation of the binding of human growth hormone to the human prolactin receptor.

Human growth hormone (hGH) elicits a diverse set of biological activities including lactation that derives from binding to the prolactin (PRL) receptor. The binding affinity of hGH for the extracellular binding domain of the hPRL receptor (hPRLbp) was increased about 8000-fold by addition of 50 micromolar ZnCl2. Zinc was not required for binding of hGH to the hGH binding protein (hGHbp) or for binding of hPRL to the hPRLbp. Other divalent metal ions (Ca2+, Mg2+, Cu2+, Mn2+, and Co2+) at physiological concentrations did not support such strong binding. Scatchard analysis indicated a stoichiometry of one Zn2+ per hGH.hPRLbp complex. Mutational analysis showed that a cluster of three residues (His18, His21, and Glu174) in hGH and His188 from the hPRLbp (conserved in all PRL receptors but not GH receptors) are probable Zn2+ ligands. This polypeptide hormone.receptor "zinc sandwich" provides a molecular mechanism to explain why nonprimate GHs are not lactogenic and offers a molecular link between zinc deficiency and its association with altered functions of hGH.

Comparison of pQCT parameters between ulna and radius in retired elite gymnasts: the skeletal benefits associated with long-term gymnastics are bone- and site-specific.

OBJECTIVES: To compare the skeletal benefits associated with gymnastics between ulna and radius. METHODS: 19 retired artistic gymnasts, aged 18-36 years, were compared to 24 sedentary women. Bone mineral content (BMC), total and cortical bone area (ToA, CoA), trabecular and cortical volumetric density (TrD, CoD) and cortical thickness (CoTh) were measured by pQCT at the 4% and 66% forearm. RESULTS: At the 4% site, BMC and ToA were more than twice greater at the radius than ulna whereas at the 66% site, BMC, ToA, CoA, CoTh and SSIpol were 20 to 51% greater at the ulna than radius in both groups (p<0.0001). At the 4% site, the skeletal benefits in BMC of the retired gymnasts over the non-gymnasts were 1.9 times greater at the radius than ulna (p<0.001), with enlarged bone size at the distal radius only. In contrast, the skeletal benefits at the 66% site were twice greater at the ulna than radius for BMC and CoA (p<0.01). CONCLUSION: Whereas the skeletal benefits associated with long-term gymnastics were greater at the radius than ulna in the distal forearm, the reverse was found in the proximal forearm, suggesting both bones should be analysed when investigating forearm strength.

Relationship between indices of adiposity obtained by peripheral quantitative computed tomography and dual-energy X-ray absorptiometry in pre-pubertal children.

BACKGROUND/AIM: The study investigated the relationship between indices of adiposity measured by peripheral quantitative computed tomography (pQCT) and dual-energy X-ray absorptiometry (DXA) in pre-pubertal children. SUBJECTS AND METHODS: DXA-derived per cent body fat (%BF) was measured in 284 boys and 288 girls, aged 7-10 years. Cross-sections of the forearm (n=427) and lower leg (n=560) were obtained by pQCT to measure total cross-sectional area of the limb (Total CSA), Muscle CSA, Fat CSA, %Fat CSA (Fat CSA/Total CSAx100) and muscle density. RESULTS: Peripheral QCT-derived %Fat CSA in the forearm and lower leg correlated strongly with DXA-derived %BF (r=0.83-0.89, p<0.01) in both boys and girls. However, forearm and lower leg %Fat CSA were higher than whole body %BF by 5% and 10%, respectively. A better prediction of whole-body %BF was achieved by including %Fat CSA, muscle density and height into a hierarchical regression model. Using sex-specific regression equations, 87.7% of the boys and 83.7% of the girls had a predicted %BF within 3% units of the %BF obtained by DXA. CONCLUSION: In pre-pubertal children, pQCT measures of adiposity are strongly associated with whole-body per cent body fat. This reproducible method could be an alternative technique to estimate body composition in this population.

Adiposity and tendinopathy.

PURPOSE: Tendon injuries (tendinopathy) are prevalent across the population, affecting active and inactive individuals and manual workers. The aetiology of tendinopathy is not known. However, extrinsic factors such as load are known to affect the prevalence. More recently, intrinsic factors have been shown to also affect tendons; genes, biomechanics, and strength have been shown to influence tendon disease. One intrinsic factor that appears to have an association with tendinopathy is body composition; more specifically central adiposity. Several studies have reported this association, and several studies have found the association when reporting other aspects of tendinopathy. METHOD: This paper will detail what is known about the association between tendinopathy and body composition, examine the strength of the association by evaluating studies in the area and speculate on potential mechanisms for the association. RESULTS: The association between tendon health and adiposity, especially central adiposity, warrants further investigation. CONCLUSION: There may be an interaction between adiposity and tendon pathology. Adiposity may be a key intrinsic risk factor that is translated into tendon disease in the presence of additional intrinsic (e.g., diabetes) and extrinsic factors (e.g., load).

The differing tempo of growth in bone size, mass, and density in girls is region-specific.

The differing tempo and direction of growth of the periosteal and endocortical surfaces, and the differing tempo of growth of the axial and appendicular skeleton, may predispose to regional deficits in bone size, bone mineral content (BMC), and volumetric bone mineral density (vBMD). These traits were measured during 2 years by dual x-ray absorptiometry in 109 girls. By 7 years of age, bone size was approximately 80% of its maturational peak, and BMC was approximately 40% of its peak. Before puberty, the legs grew more rapidly than the trunk. During puberty, the growth spurt was truncal. Between 7 and 17 years, femoral and lumbar spine BMC increased by 50-150% because bone size increased. vBMD increased by 10-30%. Thus, growth builds a bigger, but only moderately denser, skeleton. Regions growing rapidly, or distant from their peak, may be more severely affected by illness than those growing slowly or nearer completion of growth. Depending on the age of exposure to disease, deficits may occur in limb dimensions (prepuberty), spine dimensions (early puberty), or vBMD by interference with mineral accrual (late puberty). As vBMD is independent of age before puberty, the position of an individual's vBMD in the population distribution is established early in life. Bone fragility in old age may have its foundations in growth.

Longitudinal broadening of quenched jets in turbulent color fields.

The nearside distribution of particles at intermediate transverse momentum, associated with a high momentum trigger hadron produced in a high energy heavy-ion collision, is broadened in rapidity compared with the jet cone. This broadened distribution is thought to contain the energy lost by the progenitor parton of the trigger hadron. We show that the broadening can be explained as the final-state deflection of the gluons radiated from the hard parton inside the medium by soft, transversely oriented, turbulent color fields that arise in the presence of plasma instabilities. The magnitude of the effect is found to grow with medium size and density and diminish with increasing energy of the associated hadron.

The primacy of NF1 loss as the driver of tumorigenesis in neurofibromatosis type 1-associated plexiform neurofibromas.

Neurofibromatosis type 1 (NF1) is a common tumor-predisposition disorder due to germline mutations in the tumor suppressor gene NF1. A virtually pathognomonic finding of NF1 is the plexiform neurofibroma (PN), a benign, likely congenital tumor that arises from bi-allelic inactivation of NF1. PN can undergo transformation to a malignant peripheral nerve sheath tumor, an aggressive soft-tissue sarcoma. To better understand the non-NF1 genetic contributions to PN pathogenesis, we performed whole-exome sequencing, RNASeq profiling and genome-wide copy-number determination for 23 low-passage Schwann cell cultures established from surgical PN material with matching germline DNA. All resected tumors were derived from routine debulking surgeries. None of the tumors were considered at risk for malignant transformation at the time; for example, there was no pain or rapid growth. Deep (~500X) NF1 exon sequencing was also conducted on tumor DNA. Non-NF1 somatic mutation verification was performed using the Ampliseq/IonTorrent platform. We identified 100% of the germline NF1 mutations and found somatic NF1 inactivation in 74% of the PN. One individual with three PNs had different NF1 somatic mutations in each tumor. The median number of somatic mutations per sample, including NF1, was one (range 0-8). NF1 was the only gene that was recurrently somatically inactivated in multiple tumors. Gene Set Enrichment Analysis of transcriptome-wide tumor RNA sequencing identified five significant (FDR<0.01) and seven trending (0.01⩽FDR<0.02) gene sets related to DNA replication, telomere maintenance and elongation, cell cycle progression, signal transduction and cell proliferation. We found no recurrent non-NF1 locus copy-number variation in PN. This is the first multi-sample whole-exome and whole-transcriptome sequencing study of NF1-associated PN. Taken together with concurrent copy-number data, our comprehensive genetic analysis reveals the primacy of NF1 loss as the driver of PN tumorigenesis.

Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39.

The genome sequences of Chlamydia trachomatis mouse pneumonitis (MoPn) strain Nigg (1 069 412 nt) and Chlamydia pneumoniae strain AR39 (1 229 853 nt) were determined using a random shotgun strategy. The MoPn genome exhibited a general conservation of gene order and content with the previously sequenced C.trachomatis serovar D. Differences between C.trachomatis strains were focused on an approximately 50 kb 'plasticity zone' near the termination origins. In this region MoPn contained three copies of a novel gene encoding a >3000 amino acid toxin homologous to a predicted toxin from Escherichia coli O157:H7 but had apparently lost the tryptophan biosyntheis genes found in serovar D in this region. The C. pneumoniae AR39 chromosome was >99.9% identical to the previously sequenced C.pneumoniae CWL029 genome, however, comparative analysis identified an invertible DNA segment upstream of the uridine kinase gene which was in different orientations in the two genomes. AR39 also contained a novel 4524 nt circular single-stranded (ss)DNA bacteriophage, the first time a virus has been reported infecting C. pneumoniae. Although the chlamydial genomes were highly conserved, there were intriguing differences in key nucleotide salvage pathways: C.pneumoniae has a uridine kinase gene for dUTP production, MoPn has a uracil phosphororibosyl transferase, while C.trachomatis serovar D contains neither gene. Chromosomal comparison revealed that there had been multiple large inversion events since the species divergence of C.trachomatis and C.pneumoniae, apparently oriented around the axis of the origin of replication and the termination region. The striking synteny of the Chlamydia genomes and prevalence of tandemly duplicated genes are evidence of minimal chromosome rearrangement and foreign gene uptake, presumably owing to the ecological isolation of the obligate intracellular parasites. In the absence of genetic analysis, comparative genomics will continue to provide insight into the virulence mechanisms of these important human pathogens.

Specificity of insertion of IS1.

A systematic study of the specificity of insertion of the transposable element IS1 into small defined-sequence plasmids (pBR322 and derivatives) was conducted to determine the features of the DNA sequence that influence target site selection. We have physically mapped several collections of independent insertions of IS1 into these plasmids and have determined: (1) that about 80% of all insertions occur in the DNA segment (about 200 base-pairs) between the unique EcoRI site of pBR322 and the beginning of the beta-lactamase gene, one of the two regions of high A + T density in this plasmid; (2) that there is a strong orientation effect in this region (almost all IS1 insertions are in one orientation) that depends on both the pBR322 sequence and the environment of the transposon in the donor molecule; and (3) that the orientation effect does not depend on the strong transcription that is directed through this region in pBR322. Furthermore, we have found that insertion of a poly(dA X dT) segment into pBR322 creates an artificial hotspot for IS1 insertion, even though it is not as attractive for insertion as the above-mentioned major hotspot. Our observations suggest that an interplay between several properties of the target sequences and the sequence environment of the donor transposon is responsible for the observed specificity of position and orientation. One of the possibilities discussed here is that preferred "entry-sites", or "signal" sequences, for the transposition complex play a major role in determining the positions and orientations of IS1 insertions.