BACKGROUND & AIMS: -2

背景与目标： -2

BACKGROUND & AIMS: :Wild-type (WT) sequence p53 peptides are attractive candidates for broadly applicable cancer vaccines. The aim of this study was to evaluate the potential of a WT p53-based immunotherapeutic approach for patients with hepatocellular carcinoma (HCC). Circulating CD8+ T cells specific for WT p53(149-157) and WT p53(264-272) HLA-A*0201 restricted epitopes were directly identified in the peripheral blood by the use of peptide/HLA-A2.1 tetramers in 24 HCC patients. Cytotoxic T lymphocyte (CTL) activity after WT p53 peptide-specific stimulation was assessed by analysis of granzyme B and interferon-gamma mRNA transcription, using a quantitative real-time polymerase chain reaction assay. Tumor immunophenotyping was performed to evaluate the p53 status, the expression of major histocompatibility complex (MHC) and costimulatory molecules in freshly isolated tumor cells. HCC patients exhibited significantly higher frequencies of WT p53-specific memory CD8+ T cells and stronger WT p53-specific CTL activity, when compared with healthy controls. Increased frequencies of p53-specific CD8+ T cells and their activity correlated with selective HLA-A2 allele loss and reduced costimulatory molecule expression of tumor cells. Moreover, augmented numbers of p53-specific T cells coincided with high MHC class II expression in tumor cells but were inversely related to the T status of the tumor node metastasis staging system. Our results indicate the existence of natural immunosurveillance and tumor immune evasion, involving a T cell response against WT p53 tumor antigen in patients with HCC. These findings may have important implications for the future development of cancer vaccines.

背景与目标： : 野生型 (WT) 序列p53肽是广泛适用的癌症疫苗的有吸引力的候选者。这项研究的目的是评估WT p53-based免疫治疗方法对肝细胞癌 (HCC) 患者的潜力。通过在24例HCC患者中使用肽/HLA-A2.1四聚体，在外周血中直接鉴定了对WT p53(149-157) 和WT p53(264-272) hla-a * 0201限制性表位特异性的循环CD8 + T细胞。使用定量实时聚合酶链反应测定法，通过分析颗粒酶B和干扰素-γ mRNA转录来评估WT p53肽特异性刺激后的细胞毒性T淋巴细胞 (CTL) 活性。进行肿瘤免疫表型分析以评估新鲜分离的肿瘤细胞中的p53状态，主要组织相容性复合物 (MHC) 和共刺激分子的表达。与健康对照组相比，HCC患者表现出明显更高的WT p53-specific记忆CD8 + T细胞频率和更强的WT p53-specific CTL活性。p53-specific CD8 + T细胞的频率增加及其活性与肿瘤细胞的选择性HLA-A2等位基因丢失和共刺激分子表达降低相关。此外，p53-specific T细胞数量的增加与肿瘤细胞中高MHC II类表达相吻合，但与肿瘤淋巴结转移分期系统的T状态成反比。我们的结果表明存在自然免疫监视和肿瘤免疫逃避，涉及HCC患者针对WT p53肿瘤抗原的T细胞反应。这些发现可能对癌症疫苗的未来发展具有重要意义。

BACKGROUND & AIMS: :A 29-year-old man was admitted to our institution 10 days after he had undergone an urgent exploratory laparotomy at a local army hospital after a terrorist bombing attack. On admission, deep second-degree and third-degree burns involving 25% of the upper and lower extremities were present, together with a 25 x 10-cm abdominal full-thickness blast injury defect on the left side, an infected eviscerated midline incision, and a colostomy on the right side of the abdomen. The patient underwent a second laparotomy, at which time the intraabdominal abscess was drained, and the abdominal cavity was irrigated with saline. A jejunal perforation was found and sutured. The abdominal cavity was left open and covered with a Bogota bag for temporary closure. On postburn day 18, the patient underwent débridment and grafting of the third-degree burns to the left and right arm and right lower extremities. After several débridment sessions (postburn days 16, 18, 20, 22, and 24), an abdominal skin release and reapproximation were performed (postburn day 26). On postburn day 36, split-thickness skin grafts were placed directly on the granulated tissue of the intestines and on a defect in the left flank and iliac regions. Postoperatively, the patient did well. He was discharged on postburn day 78 with all wounds well healed. In our opinion, temporary closure followed by direct application of meshed split-thickness skin grafts to exposed abdominal viscera represents a simple method of reconstruction that can be safely performed, with minimal risk, on critically ill patients.

背景与目标： : 一名29岁的男子在恐怖爆炸袭击后在当地一家军队医院接受紧急剖腹探查术10天后被送入我们的机构。入院时，出现了涉及上下肢25% 的深二度和三度烧伤，左侧有25x10 cm的腹部全层爆炸伤缺损，感染的内脏中线切口，以及腹部右侧的结肠造口术。患者接受了第二次剖腹手术，此时引流了腹腔内脓肿，并用盐水冲洗了腹腔。发现空肠穿孔并缝合。腹腔保持开放状态，并用波哥大袋覆盖，以暂时关闭。在烧伤后第18天，患者接受了三度烧伤的移植，并将其移植到左，右臂和右下肢。经过几次缓解 (烧伤后第16、18、20、22和24天) 后，进行了腹部皮肤释放和重新逼近 (烧伤后第26天)。在烧伤后的第36天，将厚度分开的皮肤移植物直接放置在肠的颗粒状组织以及左侧和ili区的缺损处。术后，患者表现良好。他在烧伤后第78天出院，所有伤口均愈合良好。在我们看来，暂时封闭，然后直接将网状厚度的皮肤移植物应用到暴露的腹部内脏上，代表了一种简单的重建方法，可以对危重病人安全地进行，风险最小。

BACKGROUND & AIMS: BACKGROUND:We examined loss of heterozygosity (LOH) of the P53 gene in bladder cancer, and investigated the role of the P53 gene on malignant progression of papillary tumors. In addition, the clonality of recurrent bladder cancer was examined. METHODS:LOH of the P53 gene was analyzed in 67 bladder cancers from 47 patients. DNA was extracted from formalin-fixed, paraffin-embedded tissues, amplified by the polymerase chain reaction (PCR) at 3 polymorphic loci in the P53 gene, and analyzed with nonradioisotopic single-strand conformation polymorphism (Non-RI SSCP) analysis. RESULTS:Out of 40 informative samples, LOH was detected in 13 samples, containing 4 of 7 in grade 3 (57%), 9 of 23 in grade 2 (39%), and none of 10 in grade 1 (10%). Statistical significance was observed between the LOH in grades 1 and 2, and in grades 1 and 3. An analysis of 5 cases showing malignant progression revealed that 3 (60%) showed an LOH in the primary tumor, and 2 showed LOH in recurrent tumors, in contrast to LOH found in 3 cases of 19 (16%) not showing malignant progression. Four cases with metachronous recurrence exhibited LOH; 2 at recurrent tumors, 1 only at the initial tumor, and 1 at both tumors. CONCLUSIONS:The alterations of the P53 gene were considered to correlate with tumor grade, and contribute to the malignant progression of bladder cancer. LOH in the P53 gene may serve as a clinical indicator for prognosis in superficial bladder cancer.

背景与目标：

BACKGROUND & AIMS: A 43-year-old Japanese male vagrant collapsed suddenly in a police station. The man was resuscitated in hospital but died about 13 h later. An autopsy revealed necrotizing fasciitis in the genitalia and lower abdominal wall. The man was considered to have died from endotoxic shock following on Fournier's gangrene.

背景与目标： 一名43岁的日本男性流浪者在警察局突然倒塌。该名男子在医院复苏，但约13小时后死亡。尸检显示生殖器和下腹壁坏死性筋膜炎。该男子被认为死于Fournier坏疽后的内毒素休克。

BACKGROUND & AIMS: BACKGROUND:This study explored the combined impact of depression and inflammation on memory functioning among Mexican-American adults and elders. METHODS:Data were analyzed from 381 participants of the Health and Aging Brain study among Latino Elders (HABLE). Fasting serum samples were collected and assayed in duplicate using electrochemiluminesce on the SECTOR Imager 2400A from Meso Scale Discovery. Positive DepE (depression endophenotype) was codified as any score >1 on a five-point scale based on the GDS-30. Inflammation was determined by TNFα levels and categorized by tertiles (1st, 2nd, 3rd). WMS-III LMI and LMII as well as CERAD were utilized as measures of memory. ANOVAs examined group differences between positive DepE and inflammation tertiles with neuropsychological scale scores as outcome variables. Logistic regressions were used to examine level of inflammation and DepE positive status on the risk for MCI. RESULTS:Positive DepE as well as higher inflammation were both independently found to be associated with lower memory scores. Among DepE positive, those who were high in inflammation (3rd tertile) were found to perform significantly worse on WMS-III LM I (F = 4.75, p = 0.003), WMS-III LM II (F = 8.18, p < 0.001), and CERAD List Learning (F = 17.37, p < 0.001) when compared to those low on inflammation (1st tertile). The combination of DepE positive and highest tertile of inflammation was associated with increased risk for MCI diagnosis (OR = 6.06; 95% CI = 3.9-11.2, p < 0.001). CONCLUSION:Presence of elevated inflammation and positive DepE scores increased risk for worse memory among Mexican-American older adults. Additionally, the combination of DepE and high inflammation was associated with increased risk for MCI diagnosis. This work suggests that depression and inflammation are independently associated with worse memory among Mexican-American adults and elders; however, the combination of both increases risk for poorer memory beyond either alone.

背景与目标：

BACKGROUND & AIMS: OBJECTIVES:Monochorionic (MC) twins are at increased risk of early fetal loss secondary to vascular complications such as twin-twin transfusion syndrome (TTTS). This study compared the early perinatal loss rates between MC and dichorionic (DC) twins in an era of invasive treatment for TTTS. METHODS:This was a retrospective study of all twin pregnancies of known chorionicity from a large regional cohort of nine hospitals over a 10-year period. Ultrasound data were matched to hospital delivery records and to a mandatory national register of pregnancy losses. Prospective risk of pregnancy loss from 14 to 24 weeks' gestation was calculated and the survival trend of MC and DC twins was analyzed using Kaplan-Meier survival analysis. RESULTS:The analysis included 3117 twin pregnancies (605 MC and 2512 DC). The total risk of early pregnancy loss (miscarriage and neonatal death) before 24 weeks was significantly higher in MC twins (60.3 per 1000 fetuses) than in DC twins (6.6 per 1000 fetuses), with a relative risk of 9.18 (95% CI, 6.0-13.9). Survival analysis showed a significant difference in overall and early mortality between MC and DC twins (log-rank test, P < 0.0001), while no difference was noted after 24 weeks' gestation (log-rank test, P = 0.08). CONCLUSIONS:Early pregnancy loss is significantly more common in MC than in DC twins, but no difference in the prospective risk of mortality between MC and DC twins is evident after 24 weeks' gestation. The observed early mortality rate has almost halved in comparison with previous studies in the published literature. Early detection and prompt treatment of complications in MC twins are likely to have contributed to this improvement in outcome.

背景与目标：

BACKGROUND & AIMS: :Triple-negative breast cancer is characterized by aggressive tumours whose cells lack oestrogen and progesterone receptors and do not over-express HER2. It accounts for approximately 10-15% of breast cancer cases. We sought to generate a cellular model of chemotherapy drug resistance for this type of disease to provide the tools for the development of new therapies. Doxorubicin is a component of some chemotherapy regimes used to treat this form of cancer but resistance preventing disease eradication frequently occurs, mainly due to over-expression of drug transporters such as P-glycoprotein. CALDOX cells were generated by exposure of CAL51 to doxorubicin. Resistance to doxorubicin did not involve drug transporters, as the both parental and resistant cells accumulated doxorubicin to comparable levels. CALDOX cells had slower proliferation rate and an extended G1 cell cycle stage than the parental line, mainly due to an intrinsic activation of CDNK1 (p21), but this cell cycle block was not involved in the mechanism of resistance. CALDOX cells had reduced levels of TOP2A (topoisomerase IIα) and were cross resistant to the topoisomerase II inhibitors etoposide and mitoxantrone. CALDOX cells showed collateral sensitivity to carmustine due to the lack of O⁶-methylguanine-DNA-methyltransferase (MGMT) expression, related to the hypermethylation of its promoter. The collateral sensitivity of CALDOX cells to carmustine provides the rationale to evaluate MGMT promoter methylation status to design better therapeutic strategies for triple negative breast cancer.

背景与目标： : 三阴性乳腺癌的特征是侵袭性肿瘤，其细胞缺乏雌激素和孕激素受体，并且不会过度表达her2。它约占乳腺癌病例的10-15%。我们试图为这种类型的疾病生成化学疗法耐药性的细胞模型，以为开发新疗法提供工具。阿霉素是用于治疗这种癌症的某些化学疗法方案的组成部分，但经常发生抗药性预防疾病根除，这主要是由于药物转运蛋白 (例如P-糖蛋白) 的过表达。CALDOX细胞是通过将CAL51暴露于阿霉素而产生的。对阿霉素的耐药性不涉及药物转运蛋白，因为亲本和耐药细胞都积累了阿霉素的水平相当。与亲本系相比，钙氧化细胞的增殖速度较慢，G1细胞周期阶段延长，这主要是由于CDNK1 (p21) 的内在激活，但这种细胞周期阻滞不参与抗性机制。钙氧化细胞的TOP2A (拓扑异构酶II α) 水平降低，并且对拓扑异构酶II抑制剂依托泊苷和米托蒽醌具有交叉抗性。由于缺乏o-甲基鸟嘌呤-DNA-甲基转移酶 (MGMT) 表达，CALDOX细胞对卡莫司汀表现出附带敏感性，这与其启动子的高甲基化有关。CALDOX细胞对卡莫司汀的附带敏感性为评估MGMT启动子甲基化状态以设计更好的三阴性乳腺癌治疗策略提供了依据。

BACKGROUND & AIMS: :Polybrominated diphenyl ethers (PBDEs), chemicals commonly used as flame-retardants in consumer products, are emerging persistent organic pollutants that are ubiquitous in the environment. In this study, we report a PBDE-respiring isolate - Dehalococcoides mccartyi strain GY50, which debrominates the most toxic tetra- and penta-BDE congeners (∼1.4 µM) to diphenyl ether within 12 days with hydrogen as the electron donor. The complete genome sequence revealed 26 reductive dehalogenase homologous genes (rdhAs), among which three genes (pbrA1, pbrA2 and pbrA3) were highly expressed during PBDE debromination. After 10 transfers of GY50 with trichloroethene or 2,4,6-trichlorophenol as the electron acceptor instead of PBDEs, the ssrA-specific genome island (ssrA-GI) containing pbrA1 and pbrA2 was deleted from the genome of strain GY50, leading to two variants (strain GY52 with trichloroethene, strain GY55 with 2,4,6-trichlorophenol) with identically impaired debromination capabilities (debromination of penta-/tetra-BDEs ceased at di-BDE 15). Through analysis of Illumina paired-end sequencing data, we identified read pairs that probably came from variants that contain ssrA-GI deletions, indicating their possible presence in the original strain GY50 culture. The two variant strains provide real-time examples on rapid evolution of organohalide-respiring organisms. As PBDE-respiring organisms, GY50-like strains may serve as key players in detoxifying PBDEs in contaminated environments.

背景与目标： : 多溴联苯醚 (PBDEs) 是消费品中常用作阻燃剂的化学品，是环境中普遍存在的新兴持久性有机污染物。在这项研究中，我们报告了一个PBDE呼吸分离物-Dehalococcoides mccartyi菌株GY50，该菌株在12天内以氢为电子供体将毒性最大的四溴化和五溴化同系物 (〜1.4 µ m) 脱溴为二苯醚。完整的基因组序列揭示了26个还原脱卤酶同源基因 (rdha)，其中三个基因 (pbrA1，pbrA2和pbrA3) 在PBDE脱溴过程中高度表达。用三氯乙烯或2,4，6-三氯苯酚作为电子受体而不是多溴二苯醚对GY50进行10次转移后，从菌株GY50的基因组中删除了含有pbrA1和pbrA2的ssrA特异性基因组岛 (ssrA-GI)，导致两个变体 (具有三氯乙烯的菌株GY52，具有2,4的菌株GY55，6-三氯苯酚) 的脱溴能力同样受损 (五溴/四溴二苯醚的脱溴在二溴二苯醚15时停止)。通过对Illumina配对末端测序数据的分析，我们确定了可能来自包含ssrA-GI缺失的变体的读取对，表明它们可能存在于原始菌株GY50培养物中。这两个变体菌株提供了有关有机卤化物呼吸生物快速进化的实时示例。作为多溴二苯醚呼吸的生物，GY50-like菌株可能是污染环境中多溴二苯醚解毒的关键角色。

BACKGROUND & AIMS: OBJECTIVE:Unidentified hearing loss at birth can adversely affect speech and language development as well as academic achievement and social-emotional development. Historically, moderate-to-severe hearing loss in young children was not detected until well beyond the newborn period. Around 0.5 to 5 in every 1000 neonates and infants have congenital or early childhood onset sensorineural hearing impairment. When identification and intervention occur at no later than 6 months of age, the infants perform much higher on school-related measures. Therefore, early detection is vitally important. Toward the end of 2009, the Israeli ministry of health issued a directive establishing a universal newborn hearing screening program in all hospitals in the country from 01.01.10. The objectives of this study are to evaluate a newly established universal newborn hearing screening program, to assess performance and to compare measurements of performance to performance benchmarks representing a consensus of expert opinion. The benchmarks are the minimal requirements that should be attained by high-quality early hearing detection programs. METHODS:As specified by the ministry of health, a two-stage screening protocol was implemented using otoacoustic emissions and automated auditory brainstem response. Screening results of all neonates born from the initiation of the program on 15th March 2010 until the end of 2011 were reviewed. RESULTS:The total number of live births during the study period was 5496. Of these, 5334 (97%) started screening for hearing loss but only 5212 completed the screening process, giving a screening coverage of 94.8%. Of the 5212 neonates completing the screening process, 270 (5.18%) were referred for full diagnostic testing. CONCLUSIONS:The newly established universal newborn hearing screening program at the Ziv Medical Center in Zefat closely approaches, but does not yet meet the minimal requirements that should be attained by high-quality early hearing detection programs. Every effort should be made to complete the screening tests before discharge from hospital. Screening staff should actively encourage parents to participate in all stages of early detection.

背景与目标：

BACKGROUND & AIMS: BACKGROUND:Reductions in admissions for myocardial infarction (MI) have been reported in locales where smoke-free workplace laws have been implemented, but no study has assessed sudden cardiac death in that setting. In 2002, a smoke-free restaurant ordinance was implemented in Olmsted County, Minnesota, and in 2007, all workplaces, including bars, became smoke free. METHODS:To evaluate the population impact of smoke-free laws, we measured, through the Rochester Epidemiology Project, the incidence of MI and sudden cardiac death in Olmsted County during the 18-month period before and after implementation of each smoke-free ordinance. All MIs were continuously abstracted and validated, using rigorous standardized criteria relying on biomarkers, cardiac pain, and Minnesota coding of the electrocardiogram. Sudden cardiac death was defined as out-of-hospital deaths associated with coronary disease. RESULTS:Comparing the 18 months before implementation of the smoke-free restaurant ordinance with the 18 months after implementation of the smoke-free workplace law, the incidence of MI declined by 33% (P < .001), from 150.8 to 100.7 per 100,000 population, and the incidence of sudden cardiac death declined by 17% (P = .13), from 109.1 to 92.0 per 100,000 population. During the same period, the prevalence of smoking declined and that of hypertension, diabetes mellitus, hypercholesterolemia, and obesity either remained constant or increased. CONCLUSIONS:A substantial decline in the incidence of MI was observed after smoke-free laws were implemented, the magnitude of which is not explained by community cointerventions or changes in cardiovascular risk factors with the exception of smoking prevalence. As trends in other risk factors do not appear explanatory, smoke-free workplace laws seem to be ecologically related to these favorable trends. Secondhand smoke exposure should be considered a modifiable risk factor for MI. All people should avoid secondhand smoke to the extent possible, and people with coronary heart disease should have no exposure to secondhand smoke.

背景与目标：

BACKGROUND & AIMS: JUSTIFICATION:Hearing impairment is one of the most critical sensory impairments with significant social and psychological consequences. Evidence-based, standardized national guidelines are needed for professionals to screen for hearing impairment during the neonatal period. PROCESS:The meeting on formulation of national consensus guidelines on developmental disorders was organized by Indian Academy of Pediatrics in Mumbai, on 18th and 19th December, 2015. The invited experts included Pediatricians, Developmental Pediatricians, Pediatric Neurologists and Clinical Psychologists. The participants framed guidelines after extensive discussions. OBJECTIVE:To provide guidelines on newborn hearing screening in India. RECOMMENDATIONS:The first screening should be conducted before the neonate's discharge from the hospital - if it 'fails', then it should be repeated after four weeks, or at first immunization visit. If it 'fails' again, then Auditory Brainstem Response (ABR) audiometry should be conducted. All babies admitted to intensive care unit should be screened via ABR. All babies with abnormal ABR should undergo detailed evaluation, hearing aid fitting and auditory rehabilitation, before six months of age. The goal is to screen newborn babies before one month of age, diagnose hearing loss before three months of age and start intervention before six months of age.

背景与目标：

BACKGROUND & AIMS: OBJECTIVE:Although there is evidence for a beneficial effect of increased quadriceps strength on knee symptoms, the effect on knee structure is unclear. We undertook this study to examine the relationship between change in vastus medialis cross-sectional area (CSA) and knee pain, tibial cartilage volume, and risk of knee replacement in subjects with symptomatic knee osteoarthritis (OA). METHODS:One hundred seventeen subjects with symptomatic knee OA underwent magnetic resonance imaging of the knee at baseline and at 2 and 4.5 years. Vastus medialis CSA was measured at baseline and at 2 years. Tibial cartilage volume was measured at baseline and at 2 and 4.5 years. Knee pain was assessed by the Western Ontario and McMaster Universities Osteoarthritis Index at baseline and at 2 years. The frequency of knee joint replacement over 4 years was determined. Regression coefficients (B) and odds ratios were determined along with 95% confidence intervals (95% CIs). RESULTS:After adjusting for confounders, baseline vastus medialis CSA was inversely associated with current knee pain (r = -0.16, P = 0.04) and with medial tibial cartilage volume loss from baseline to 2 years (B coefficient -10.9 [95% CI -19.5, -2.3]), but not with baseline tibial cartilage volume. In addition, an increase in vastus medialis CSA from baseline to 2 years was associated with reduced knee pain over the same time period (r = 0.24, P = 0.007), reduced medial tibial cartilage loss from 2 to 4.5 years (B coefficient -16.8 [95% CI -28.9, -4.6]), and reduced risk of knee replacement over 4 years (odds ratio 0.61 [95% CI 0.40, 0.94]). CONCLUSION:In a population of patients with symptomatic knee OA, increased vastus medialis size was associated with reduced knee pain and beneficial structural changes at the knee, suggesting that management of knee pain and optimizing vastus medialis size are important in reducing OA progression and subsequent knee replacement.

背景与目标：

BACKGROUND & AIMS: BACKGROUND:In Xenopus the bone morphogenetic protein growth and differentiation factor 6 (GDF6) is expressed at the edge of the neural plate, and within the anterior neural plate including the eye fields. Here we address the role of GDF6 in neural and eye development by morpholino knockdown experiments. RESULTS:We show that depletion of GDF6 (BMP13) resulted in a reduction in eye size, loss of laminar structure and a reduction in differentiated neural cell types within the retina. This correlated with a reduction in staining for Smad1/5/8 phosphorylation indicating a decrease in GDF6 signalling through loss of phosphorylation of these intracellular mediators of bone morphogenetic protein (BMP) signalling. In addition, the Pax6 expression domain is reduced in size at early optic vesicle stages. Neural cell adhesion molecule (NCAM) is generally reduced in intensity along the neural tube, while in the retina and brain discreet patches of NCAM expression are also lost. GDF6 knock down resulted in an increase in cell death along the neural tube and within the retina as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. CONCLUSION:Our data demonstrate that GDF6 has an important role in neural differentiation in the eye as well as within the central nervous system, and that GDF6 may act in some way to maintain cell survival within the ectoderm, during the normal waves of programmed cell death.

背景与目标：

BACKGROUND & AIMS: The effects of medium composition, nutrient limitation and dilution rate on the loss of the recombinant plasmid pLG669-z and plasmid-borne beta-galactosidase expression were studied in batch and chemostat cultures of Saccharomyces cerevisiae strain CGpLG. The difference in growth rates between plasmid-free and plasmid-containing cells (delta mu) and the rate of segregation (R) were determined and some common factors resulting from the effect of medium composition on plasmid loss were identified. Glucose-limited chemostat cultures of CGpLG grown on defined medium were more stable at higher dilution rates and exhibited delta mu-dominated plasmid loss kinetics. Similar cultures grown on complex medium were more stable at lower dilution rates and exhibited R-dominated plasmid loss kinetics. Overall plasmid stability was greatest in phosphate-limited chemostat cultures grown on defined medium and was least stable in magnesium-limited cultures grown on defined medium. delta mu decreased and R increased with increased dilution rate, irrespective of medium composition. Increased plasmid loss rates at high or low dilution rates would appear to be characteristic of loss kinetics dominated by R or delta mu, respectively. Growth of glucose-limited chemostat cultures on complex medium decreased delta mu values but increased R values, in comparison to those cultures grown on defined medium. Any increased stability that a complex medium-induced reduction of delta mu may have conferred was counteracted by an increased R value. Increased beta-galactosidase productivity was correlated with increased plasmid stability only in glucose-limited chemostat cultures grown on defined medium and not in those grown on complex medium. Previous studies have yielded contrasting responses with regard to the effect of dilution rate on recombinant plasmid loss from S. cerevisiae. Our findings can account for these differences and may be generally valid for the stability of similar yeast plasmid constructs. This information would facilitate the design of bioprocesses, where recombinant plasmid instability results in reduced culture productivity.

背景与目标： 在酿酒酵母菌株CGpLG的分批和恒化器培养中，研究了培养基组成，营养限制和稀释率对重组质粒pLG669-z丢失和质粒传播的 β-半乳糖苷酶表达的影响。确定了无质粒和含质粒细胞之间的生长速率差异 (delta mu) 和分离速率 (R)，并确定了由培养基组成对质粒丢失的影响引起的一些常见因素。在确定的培养基上生长的CGpLG的葡萄糖限制的恒化器培养物在较高的稀释率下更稳定，并表现出delta mu主导的质粒丢失动力学。在复杂培养基上生长的类似培养物在较低的稀释率下更稳定，并表现出R主导的质粒丢失动力学。在限定培养基上生长的磷酸盐限制的恒化器培养物中，总体质粒稳定性最高，而在限定培养基上生长的镁限制的培养物中，质粒稳定性最低。与培养基组成无关，delta mu降低，R随着稀释率的增加而增加。在高或低稀释率下增加的质粒丢失率似乎分别是R或delta mu主导的损失动力学的特征。与在确定的培养基上生长的那些培养物相比，在复杂培养基上生长的葡萄糖限制的恒化器培养物的生长降低了delta mu值，但增加了r值。复杂的培养基诱导的 δ mu减少可能带来的任何增加的稳定性都被r值的增加所抵消。仅在在确定的培养基上生长的葡萄糖限制的恒化器培养物中，而不是在复杂培养基上生长的那些中，β-半乳糖苷酶生产率的提高与质粒稳定性的提高相关。先前的研究在稀释率对酿酒酵母重组质粒丢失的影响方面产生了相反的反应。我们的发现可以解释这些差异，并且通常对于类似酵母质粒构建体的稳定性有效。此信息将有助于生物过程的设计，其中重组质粒的不稳定性导致培养生产率降低。