Immediate Adjuvant Chemotherapy in Non-Metastatic Colon Cancer: Phase I Trial Evaluating a Novel Treatment Protocol

BackgroundThe optimal timing of adjuvant chemotherapy (AC) in non-metastatic colon cancer is poorly defined. Delays in AC result in decreased survival. Effective cytotoxic treatments should be considered during the perioperative phase of care. The immediate adjuvant chemotherapy (IAC) concept intends to capitalize on the therapeutic benefits that can be achieved in the perioperative period. We aim to demonstrate that IAC is safe and tolerable.Patient and MethodsMicrosatellite stable invasive adenocarcinomas were treated with intravenous Leucovorin 20 mg/m2 and single dose of 5-Flurouracil 400mg/m2 at the time of surgery. High-risk stage II and stage III received the first dose of standard AC at 14 days after surgery. Serial measurements of blood-based biomarkers were measured. Quality of life (QOL) was measured using EORTC QLQ-C30.ResultsOf the 20 patients recruited, 40% had final pathology of stage III, 40% stage II and 20% stage I. All patients received intra-operative chemotherapy with no associated morbidity. Median length of stay was 2 days (range of 2-4). There was no intraoperative morbidity with 5% (N = 1) grade 3 complication. AC was administered to 65% of patients. The median time to AC was 14 days (range 14-36). Overall quality of life and health scores were similar before surgery and at 30-day postoperatively (P < .05).ConclusionsA protocol based on IAC starting at the time of surgical resection was found to be safe and feasible with no adverse effects on surgical morbidity or quality of life. Further prospective studies are needed to explore the oncologic benefit of this novel systemic treatment approach.     

Sleep duration and daytime sleepiness in a large sample of Brazilian high school adolescents

ObjectiveTo investigate the prevalence and main factors associated with short and long sleep duration and excessive daytime sleepiness in Brazilian adolescents.MethodsThis was a cross-sectional study of 11.525 students of both genders, aged 14–17 years, from the public high-school system. Sleep duration was assessed by self-report and <8 h of sleep per day was considered short sleep and >10 h, long sleep. Socio-demographic and behavioral factors were investigated through a purpose-built questionnaire and daytime somnolence was assessed by the Epworth sleepiness scale (ESS).ResultsThe overall prevalence of short and long sleep was 54.7% and 3.3%, respectively. Frequency of short sleep was lowest in the afternoon shift (38.2%) and highest in the morning shift (62.9%) and full-day students (70.0%). Insufficient sleep was more frequent in working (63.0%) than non-working adolescents (53.1%; p = 0.001) and among those who used their cell phone before bedtime (56.3%) compared to non-users (49.7%, p = 0.001). On average, ESS score was higher in subjects with short and long sleep (respectively, 9.7 ± 4.4 and 10.0 ± 4.5) compared to those with normal sleep duration (8.9 ± 4.2; p = 0.001).ConclusionInsufficient sleep and excessive daytime sleepiness are very common among urban high-school Brazilian adolescents. Full day and morning school shifts are associated with short sleep and daytime somnolence, suggesting later start times may have a role in reducing sleep loss in these subjects. Older age, work activity, and cell phone use before bedtime, are also risk factors for of short sleep in adolescents. Although less common, long sleep can also be associated with excessive daytime sleepiness.     

基于转录组数据分析药用大黄的密码子使用偏好性

目的研究药用大黄Rheum officinale转录组编码序列的密码子使用特点及其影响因素,为蒽醌类化合物异源合成的载体选择及药用大黄分子进化研究提供理论基础。方法利用perl程序及Codon W软件分析4733条药用大黄转录组编码序列的密码子使用偏好性。结果药用大黄转录组编码序列的GC、GC3平均含量分别为45.6%、44.73%,GC12与GC3存在显著正相关(r=0.215,P0.001);ENc-GC3中性图及偏倚性分析结果显示,大部分基因分布于远离期望曲线和平面中心点,大部分基因分布偏离期望曲线和中心点。通过基因高表达筛选密码子方法确定了29个药用大黄的最优密码子,大多数的最优密码子以U和A结尾。结论突变压力在药用大黄转录组编码序列的密码子使用偏好性形成过程这起到主要作用。     

苦豆子SaLDC密码子偏好性分析、优化及原核表达

目的研究苦豆子SaLDC密码子偏好性、优化及原核表达。方法运用EMBOSS在线程序中的CHIPS、CUSP和CodonW软件分析SaLDC密码子的偏好性,根据大肠杆菌密码子的偏好性对SaLDC密码子进行优化,并将优化后的基因命名为optSaLDC;构建重组表达载体pET?28a(+)?optSaLDC,转化大肠杆菌BL21(DE3);IPTG诱导SaLDC蛋白表达,并对表达蛋白进行质谱分析。结果SaLDC有28个密码子的RSCU值>1.00,偏好使用以A/T结尾的密码子;SaLDC的密码子使用偏好性与拟南芥最接近,与大肠杆菌差别最大。优化后的optSaLDC在大肠杆菌中最佳表达条件为IPTG浓度1.0 mmol/L,诱导温度15℃,诱导时间16 h。LC?MS/MS鉴定诱导得到的SaLDC蛋白分子质量为48990.51 Da。结论诱导获得的optSaLDC纯化蛋白可用于后续体外SaLDC酶活性分析,为深入研究该基因的功能提供依据。     

Roles of helix H69 of 23S rRNA in translation initiation

Initiation of translation involves the assembly of a ribosome complex with initiator tRNA bound to the peptidyl site and paired to the start codon of the mRNA. In bacteria, this process is kinetically controlled by three initiation factors—IF1, IF2, and IF3. Here, we show that deletion of helix H69 (∆H69) of 23S rRNA allows rapid 50S docking without concomitant IF3 release and virtually eliminates the dependence of subunit joining on start codon identity. Despite this, overall accuracy of start codon selection, based on rates of formation of elongation-competent 70S ribosomes, is largely uncompromised in the absence of H69. Thus, the fidelity function of IF3 stems primarily from its interplay with initiator tRNA rather than its anti-subunit association activity. While retaining fidelity, ∆H69 ribosomes exhibit much slower rates of overall initiation, due to the delay in IF3 release and impedance of an IF3-independent step, presumably initiator tRNA positioning. These findings clarify the roles of H69 and IF3 in the mechanism of translation initiation and explain the dominant lethal phenotype of the ∆H69 mutation.Translation initiation can be divided into two major stages in bacteria. The first stage involves assembly of the 30S initiation complex (30SIC). Facilitated by three initiation factors, initiator tRNA (N-formyl-methionyl-tRNA^fMet, or fMet-tRNA^fMet) binds to the peptidyl (P) site of the 30S subunit and pairs with the start codon on the mRNA. During the second stage, the 50S subunit associates with the 30SIC and triggers dissociation of the initiation factors, leaving the 70S initiation complex (70SIC) with fMet-tRNA^fMet in the P site, ready for elongation. Because initiation is the rate-limiting step of translation and establishes the reading frame, efficient and accurate assembly of the 70SIC is critical for cell survival.30SIC assembly can be considered a largely random-order process, although there is a preferred kinetic pathway of ligand binding. IF2 and IF3 are generally first to bind to the 30S subunit, followed by IF1 and fMet-tRNA^fMet, whereas the timing of mRNA binding depends on its sequence context and cellular concentration (1). The three initiation factors reciprocally stabilize one another in the 30SIC, and their binding induces a conformational change of the subunit, including a clockwise rotation of the head domain (2). IF1 is an 8-kDa protein that binds to helix h44, the 530 loop, and the S12 region and blocks the 30S aminoacyl (A) site (3). IF2 is a multidomain ribosome-dependent GTPase that makes extensive contacts with both the 30S subunit and fMet-tRNA^fMet. Domains G3 and C1 of IF2 bind helix h5 and h14 of 16S rRNA, the N-terminal domain (NTD) interacts with S16 and IF1, and domain C2 recognizes the acceptor stem and fMet moiety of fMet-tRNA^fMet (2, 4). These interactions contribute to functions of IF2 in increasing the on rate of fMet-tRNA^fMet binding and discriminating against elongator tRNAs (5). IF3 consists of two globular domains connected by a flexible linker (6, 7). Based on structural studies of the 30SIC, the C-terminal domain (CTD) binds to the 30S platform near helices h23, h24, and h45 of 16S rRNA (8), whereas the NTD of IF3 has been modeled to interact with the elbow region of fMet-tRNA^fMet (2). The presence of IF3 in the 30SIC induces a conformational change of IF2 and fMet-tRNA^fMet (2, 9) and increases both on and off rates of tRNA binding (5). IF1 enhances the effects of IF2 and IF3 on tRNA stability (5). Together, all three initiation factors tune the kinetics of fMet-tRNA^fMet binding for optimal efficiency and fidelity.Formation of 70SIC is highly dependent on fMet-tRNA^fMet and IF2 (10), which together provide a large surface area complementary to the 50S interface (11, 12). 50S docking stimulates the GTPase activity of IF2 (13), presumably through the interaction between the sarcin–ricin loop and IF2 G domain, and triggers a large conformational change that favors factor dissociation (12). This is accompanied by movement of the initiator tRNA into the P/P site (14). IF3 inhibits subunit joining, an effect more pronounced in the presence of noncanonical codon–anticodon base pairing (5, 15). The mechanism by which IF3 prevents spurious initiation likely involves its interplay with fMet-tRNA^fMet. IF3 and tRNA are mutually destabilizing; thus, noncanonical base pairing delays IF3 dissociation and 70SIC formation (10, 16, 17). More recently, it was shown that the conformation of IF3 in the 30SIC is also sensitive to the identity of the start codon (18).Helix H69 of 23S rRNA is a highly conserved element located at the 50S interface and interacts extensively with helix h44 of 16S rRNA through intersubunit bridge B2a (Fig. 1) (19). In the elongation complex, H69 also makes direct contact with P-site tRNA at nucleotides 11–13 and 24–25 in the D stem-loop region (20). Deletion of H69 (ΔH69, Fig. 1C) has been shown to cause dominant lethality in vivo and a severe subunit association defect in vitro that can only be partially rescued in the presence of mRNA, tRNA, and a high concentration of Mg²⁺ (21). Despite these strong effects, ΔH69 ribosomes are fully competent for in vitro poly-Phe synthesis (21) and are only mildly affected in translocation (22). During subunit association, bridge B2a is among the earliest interactions formed between the two subunits (23), and H69 is in close proximity to the binding sites of initiator tRNA and IF3 (11, 12). Thus, the interplay between H69 and these ligands may be central to the molecular mechanism of 70SIC formation.Open in a separate windowFig. 1.Location and structure of H69 in the ribosome. (A) Secondary structure of H69 and its location in the 23S rRNA [schematic diagram adapted from the Comparative RNA Website (35)]. Nucleotides making contact with P-site tRNA are highlighted in orange, regions predicted to interact with IF2 are marked with a pink line, and regions predicted to clash with IF3 are indicated by a dashed purple line. (B) Tertiary structure of H69 in the 70S ribosome [based on Protein Data Bank ID codes 2WDG and 2WDI (20)]. Atoms involved in interactions between H69 and P-site tRNA or helix h44 of 16S rRNA are shown in spheres. (C) Nucleotides replacing the native sequence (nt 1906–1930) in ΔH69, ΔLoop, and ΔLoop+4 mutants.In the current study, we investigate the effects of H69 deletion on 70SIC formation. We find that the ability of IF3 to regulate 50S docking depends largely on H69. Loss of H69 delays the release of IF3 and inhibits a subsequent conformational change of fMet-tRNA^fMet that leads to formation of elongation-competent 70SIC. Our data support a model in which H69 is critical for coupling IF3 dissociation with subunit joining as well as regulating fMet-tRNA^fMet movement during the second stage of translation initiation.     

Process Redesign to Improve First Case Surgical Starts in an Academic Institution

Purpose: On time start of the first surgical case improves operating room (OR) utilization, physician, and patient satisfaction and decreases delays in subsequent cases. The goal of our study was to evaluate the effect of a multidisciplinary initiative to improve first patient in the room (FPIR) and first case on time start (FCOTS) metrics in a tertiary care setting. Materials and Methods: A multidisciplinary committee focused on first case start data collection. Reasons for both anesthesia and surgical delays were analyzed. Improvement efforts focused on the timely completion of surgical consent, a requirement of a surgical, anesthesia, and nurse team member presence at the patient's bedside by specific time, and parallel processing in the OR. Results: Over 65,100 OR cases were analyzed between 2007 and 2014. There was a statistically significant improvement in FPIR (82.80% versus 69.60%, p < .0001) and FCOTS (66.60% versus 55.90%, p < .0001). Surgical consent completion rate increased from 35% baseline to 68%–100%, depending on the surgical subspecialty. Improvements appeared sustainable several years following process implementation for both FPIR (84.60% versus 69.60%, p < .0001) and FCOTS (67.60% versus 55.90%, p < .0001). Conclusions: Our study demonstrates a successful targeted, multidisciplinary initiative to improve first case surgical starts in an academic setting. Our approach was organizational rather than punitive or rewarding on an individual basis. Strategies included establishing concrete, time-specific goals and posting them visibly, empowering individuals to fulfill them, and ensuring no compromise in patient safety. In the complex environment of academic medicine including research protocols and teaching in the ORs, our organizational approach proved sustainable over several years.     

膜荚黄芪法呢基焦磷酸合酶基因密码子偏性分析

目的法呢基焦磷酸合酶(farnesyl pyrophosphate synthase,FPS)是调控黄芪甲苷生物合成途径关键酶,为该基因外源表达选择合适的宿主,进而为提高黄芪甲苷产量提供理论依据。方法以长白山膜荚黄芪为植物材料,克隆了FPS基因的编码序列。运用EMBOSS及Codon W等在线软件分析了FPS基因的密码子偏好性,并将其与玉米、青蒿等7种植物FPS基因及大肠杆菌基因组密码子偏好性进行比较。结果膜荚黄芪FPS基因偏好于以A或T碱基结尾的密码子,与大肠杆菌Escherichia coli基因组共有22个密码子存在差异。结论进一步提高膜荚黄芪FPS基因在大肠杆菌中的表达水平,需对其密码子进行优化。