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O6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that removes alkyl groups at the O6-position of guanine in DNA. MGMT expression is reduced or absent in many tumor types derived from a diverse range of tissues, most notably in glioma. Low MGMT expression confers significant sensitivity to DNA alkylating agents such as temozolomide, providing a natural therapeutic index over normal tissue. In this study, we sought to identify novel approaches that could maximally exploit the therapeutic index between tumor cells and normal tissues based on MGMT expression, as a means to enhance selective tumor cell killing. Temozolomide, unlike other alkylators, activated the ataxia telangiectasia and Rad3–related (ATR)–checkpoint kinase 1 (Chk1) axis in a manner that was highly dependent on MGMT status. Temozolomide induced growth delay, DNA double-strand breaks, and G2–M cell-cycle arrest, which led to ATR-dependent phosphorylation of Chk1; this effect was dependent on reduced MGMT expression. Treatment of MGMT-deficient cells with temozolomide increased sensitivity to ATR inhibitors both in vitro and in vivo across numerous tumor cell types. Taken together, this study reveals a novel approach for selectively targeting MGMT-deficient cells with ATR inhibitors and temozolomide. As ATR inhibitors are currently being tested in clinical trials, and temozolomide is a commonly used chemotherapeutic, this approach is clinically actionable. Furthermore, this interaction potently exploits a DNA-repair defect found in many cancers. Significance: Monofunctional alkylating agents sensitize MGMT-deficient tumor cells to ATR inhibitors.

Cellulose degradation is essential for the future production of many advanced biofuels. Cellulases from the filamentous fungus Trichoderma reesei are among the most efficient enzymes for the hydrolysis of cellulosic materials. One of the cellulases from T. reesei, cellobiohydrolase II (CBH2), was studied because of its industrial relevance and proven enzymatic activity. Using both crude and rigorous membrane fractionation methods we show that full length T. reesei CBH2 is exclusively localized to the outer membrane when expressed recombinantly in Escherichia coli. Even fusing signal sequence-free maltose-binding protein to the N-terminus of CBH2, which has been shown to increase solubility of other proteins, did not prevent the outer membrane localization of CBH2. These results highlight the difficulties in producing fungal cellulases in bacterial hosts and provide a stepping stone for future cellulase engineering efforts.

Death of the apical shoot and subsequent compensatory growth from previously inhibited axillary buds is a possible result of POST weed control practices. The objective of this research was to determine the efficacy of glyphosate and lactofen on ivyleaf morningglory, common waterhemp, and giant ragweed during mechanically stimulated compensatory growth. Plants were grown in the greenhouse to a height (or lateral growth) of 15 cm; at which time, the apical shoots were removed by cutting just above the cotyledonary node. Plants were allowed to regrow to 15 cm and treated with lactofen or glyphosate. Herbicide treatments were also applied to intact plants that were 15 cm in height for comparison. Weed response to herbicides during mechanically stimulated compensatory growth varied by weed species and herbicide. Giant ragweed under compensatory growth was less sensitive to both glyphosate and lactofen compared with intact plants. Comparison of GR50(the herbicide dose that reduced dry weight by 50%) values indicated that ivyleaf morningglory under compensatory growth was 1.5 times more sensitive to glyphosate than intact plants. Conversely, previously injured ivyleaf morningglory plants were less sensitive to lactofen than intact plants. The GR50for glyphosate applied to intact common waterhemp plants and plants under compensatory growth was similar. However, common waterhemp plants under compensatory growth were more sensitive to lactofen at the three lowest rates evaluated compared with intact plants. In summary, the efficacy of foliar herbicides applied to weeds that exhibit compensatory growth may be different from weeds under a normal growth state.

Field and greenhouse studies were conducted in 2002 and 2003 to evaluate mechanically stimulated compensatory growth response of ivyleaf morningglory, common waterhemp, and giant ragweed. Compensatory growth was initiated by the physical removal of the apical shoot to break apical dominance. The amount of apical shoot removed had an effect on mechanically stimulated compensatory growth of common waterhemp and giant ragweed. With these species, the more of the apical shoot removed from the plant, the less compensatory growth occurred. Removal of the shoot from above the cotyledons resulted in giant ragweed that were 48% shorter and weighed 41% less than control plants at 6 wk after shoot removal. However, the amount of apical shoot removed had no effect on the growth of ivyleaf morningglory compared with control plants at the completion of the study. The influence of plant height at the time of shoot removal on compensatory growth was specific to each weed species. Ivyleaf morningglory exhibited less compensatory growth when the plants were 10 cm at the time of shoot removal compared with 20-, 30-, and 40-cm-tall plants. Removal of the shoot when common waterhemp plants were 30 or 40 cm in height reduced plant weight by 23 and 21%, respectively, compared with control plants. However, no reductions in plant weight were observed when common waterhemp were 10 or 20 cm tall at the time of shoot removal. Giant ragweed subjected to shoot removal was smaller in most growth parameters than control plants, regardless of plant height at the time of shoot removal.

Ataxia Telangiectasia and Rad3-related (ATR) and Checkpoint kinase 1 (CHK1) stabilize stalled replication forks and prevent their collapse into DNA double strand breaks (DSBs). Inhibition of ATR in cells experiencing oncogenic stress or harboring other cancer-associated defects synergistically increases the formation of DSBs and causes synthetic lethality. Thus specific targeting of ATR represents an emerging strategy to treat a broad spectrum of cancers, most notably those that currently lack effective treatments. We and others have shown that inhibition of the ATR checkpoint kinase is synthetically lethal with multiple distinct cancer-associated mutations, including p53 loss, oncogenic stress (HRAS-G12V, KRAS-G12D, NRAS-G12D, MYC and CCNE1), deficiency in homologous recombination (BRCA1/2, PALB2, ATM loss), alternative lengthening of telomeres (ALT), chromatin modification (SETD2 loss), and others. The specificity of ATR inhibitors is vital to their successful clinical application, since off-targeting increases toxicity to normal cells and limits value in personalized treatments. We report the generation of a novel class of highly potent and specific ATR inhibitors (ATRN series) that exhibit low nanomolar activity in cultured cells (IC50 = 2-8 nM) and do not detectably inhibit ATM, DNA-PK or mTOR (IC50 > 10 µM). In side-by-side comparisons, both the potency and selectivity of the ATRN series is superior to previously reported ATR inhibitors (VE821, VE822, AZD20, AZD6738, ETP-46464). In addition, the ATRN series has sufficient bioavailability and stability for in vivo application. Our lead compound (ATRN-119) slows progression of human BRCA2-deficient PDAC (CAPAN1) and RAS oncogene-driven p53-null colon tumors in mice with minimal toxicity to tissues under normal proliferative control, including the bone marrow and intestine. Additionally, mice engrafted with BRCA2-mutant patient-derived xenograft (PDX) ovarian tumors show a significant reduction in tumor progression after 5 weeks treatment of ATRN-119, and display no toxicity or significant weight loss. In addition, cancers that maintain or reacquire HR function, and thus are resistant to treatment with PARPi or cisplatin, remain responsive to ATRN series inhibitors. Thus, ATRN-119 is highly efficacious in suppressing tumor growth in multiple murine models, including suppression of patient-derived BRCA2-mutant ovarian, and PARP resistant cancers, suggesting that the clinical application of the ATRN series will provide a new and effective treatment for human malignancies with fewer side effects than conventional chemotherapies. In summary, the ATRN series is a highly selective and potent class of ATR inhibitors with therapeutic potential for treating a broad range of cancers. The potentially identified biomarkers, in particular HR-deficiency, will inform patient selection (a critical component for delivering medical benefit) for treatment with our agent and could reduce future clinical risk and side-effects. Thus targeting patients with HR-deficient cancers is a particularly promising strategy in treating a broad range of cancers. Citation Format: Laura R. Butler, Ryan L. Ragland, Hank J. Breslin, Erin George, Tina Gill, Matthew Scheiwer, Nicolas Gordon, Karen Knudsen, Fiona Simpkins, Oren Gilad and Eric J. Brown. Potent and selective ATR inhibitors for the treatment of homologous-recombination deficient and PARPi-resistant cancers [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A16.

Ataxia Telangiectasia and Rad3-related (ATR) and Checkpoint kinase 1 (CHK1) stabilize stalled replication forks and prevent their collapse into DNA double strand breaks (DSBs). Inhibition of ATR in cells experiencing oncogenic stress or harboring other cancer-associated defects synergistically increases the formation of DSBs and causes synthetic lethality. Thus specific targeting of ATR represents an emerging strategy to treat a broad spectrum of cancers, most notably those that currently lack effective treatments. Atrin Pharmaceuticals has synthesized a novel series of small molecules that inhibit ATR at low nanomolar concentrations in cultured cells. These compounds have the highest known potency for inhibiting ATR and maintain >800-fold lower cellular activity towards other kinases of the same family (ATM, DNA-PKcs and mTOR), which are substantially off-targeted by previously reported ATR inhibitors. Atrin's lead compound (ATRN-119), as a single agent, selectively kills cancer cells subjected to oncogenic stress, alternative lengthening of telomeres (ALT) or loss of double strand break (DSB) homologous recombination repair mechanism (BRCA1 or BRCA2 deficiency). Furthermore, ATRN-119 cytotoxicity is synergistically enhanced when combined with conventional chemotherapeutics, such as etoposide, cisplatin and olaparib. Therefore, this inhibitor can be used in combination with other therapeutics to potentiate its anti-tumor activity. In vivo studies of RAS oncogene driven HCT116 p53-null flank tumors in mice demonstrate the ability of our lead compound to slow tumor progression, with minimal toxicity to tissues under normal proliferative control, including the bone marrow and intestine. Additionally, mice engrafted with BRCA2-mutant patient-derived ovarian tumors show a significant reduction in progression after 5 weeks treatment (100 mg/kg BID) of ATRN-119, and display no toxicity or significant weight loss. Thus, ATRN-119 is highly efficacious in suppressing tumor growth in multiple murine models, including suppression of patient-derived BRCA2-mutant ovarian cancer, suggesting that the clinical application of the ATRN series will provide a new and effective treatment for human malignancies with fewer side effects than conventional chemotherapies. Citation Format: Laura R. Butler, Ryan L. Ragland, Hank J. Breslin, Tina Gill, Erin George, Fiona Simpkins, Eric J. Brown, Oren Gilad. Highly specific ATR inhibitors as a therapeutic approach for a broad spectrum of cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1226.

Postoperative arrhythmias are a problem that most doctors associate with cardiothoracic surgery, but they may also complicate major abdominal surgery. The incidence depends on the type of operation and the extent of cardiac monitoring after surgery (arrhythmias are more likely to be detected in patients with continuous monitoring as opposed to intermittent monitoring). Rates ranged from 4% in a mixed cohort of patients having major general, vascular, and orthopaedic surgery to 20% in patients having elective colorectal surgery.1,2 Most of the arrhythmias are atrial in origin.1,2 Arrhythmias are associated with a longer post-operative stay and increased mortality,3,4 but few papers deal with postoperative arrhythmias, and their methodological quality is poor, with only a handful of prospective series. This contrasts with the considerable amount of work done on other complications of major abdominal surgery, such as anastomotic leakage. Most patients who develop an atrial arrhythmia after major non-cardiothoracic surgery revert to sinus rhythm, often with no anti-arrhythmic intervention.1 Indeed, atrial arrhythmias rarely cause severe haemodynamic compromise or death. It is tempting to dismiss them as transient, self limiting consequences of the physiological response to surgical trauma. However, mortality is higher in these patients because of the association with underlying complications such as myocardial infarction, respiratory failure, or sepsis in up to 80% of cases.1-3,5 The arrhythmia often resolves if the underlying cause is promptly identified and treated. Two large prospective series have reported arrhythmias in patients having non-cardiac surgery. More than 25 years ago Goldman published a prospective series of 916 patients undergoing major non-cardiac surgery, of whom 35 developed a postoperative supraventricular arrhythmia.1 In 29% of these, an acute cardiac event was thought to be the main precipitant. Importantly, from a surgeon's standpoint, major infection was thought to be the precipitant in almost as many patients (26%). Metabolic derangements such as hypokalaemia were present in 23% of the patients with arrhythmia but were identified as the principal cause of the arrhythmia in only 3%. Goldman concluded that new postoperative supraventricular arrhythmias should trigger a thorough search for remediable underlying problems. More recently, Polanczyk and colleagues published prospective data from a series of more than 4000 patients undergoing major non-cardiac surgery.3 The onset of a supraventricular arrhythmia was associated with myocardial infarction in 4% of patients, bacterial pneumonia in 7.5%, and wound infection in 8.5%. This shows that sepsis is at least as likely as an acute cardiac event or an electrolyte abnormality to be the underlying precipitant of a supraventricular arrhythmia. Although the cohort included several patients having thoracic surgery, non-vascular abdominal surgery was an independent predictor of supraventricular arrhythmia. The findings are relevant to general surgeons. Mortality from sepsis (a clinical syndrome defined by the presence of infection and a systemic inflammatory response6) and septic shock (a state of acute circulatory failure characterised by persistent arterial hypotension unexplained by other causes6) has remained static for years. The surviving sepsis campaign aims to raise awareness of the diagnosis and treatment of sepsis.7 The campaign's ultimate goal is to reduce mortality from sepsis by 25%. Early identification and intervention is essential to maximise the prospects of successful treatment. In about half of patients undergoing general surgery who develop an arrhythmia, the underlying precipitant is not identified for another 12-24 hours.5 Instead, the tendency is to focus solely on managing the arrhythmia and to neglect the search for a cause. If the precipitant is sepsis, this delay may prove crucial. Patients with a new arrhythmia are often assessed initially by junior surgical staff. In the United Kingdom, the care of the critically ill surgical patient (CCRiSP) course includes elements on the management of arrhythmia. This course is currently taken at a relatively late stage in basic surgical training, but its contents would be useful for foundation level surgical staff. In the United States, new arrhythmias after major abdominal surgery are usually managed by surgeons on the surgical intensive care unit. In the UK, surgeons usually refer patients with arrhythmias to medical doctors or anaesthetists for management. Their help may be needed for control of the heart rate or rhythm, but it is essential for the surgeon to consider, investigate, and diagnose any underlying surgical problem that may have triggered the arrhythmia. Increased awareness and education of junior surgical staff, and early involvement of senior surgical staff in the care of these patients, may avoid detrimental delay.

Contains fulltext : 186317.pdf (Publisher’s version ) (Open Access) T7-based RNA amplification is applied when there is insufficient RNA. The overall extent of amplification can be measured spectrophotometrically (i.e., quantifying RNA yields), but this measurement does not provide information about the RNA amplification of individual genes. Here we describe a method applying real-time quantitative PCR, which enables the monitoring of RNA amplification of individual genes. The amount of RNA before and after T7-based RNA amplification was determined by real-time quantitative PCR for three housekeeping genes: beta-2-microglobulin, porphobilinogen deaminase, and serine dehydratase, which are, respectively, a high, intermediate/low, and low copy transcript. Real-time quantitative PCR appeared to be suitable to determine the extent of RNA amplification, as was reflected by the low intra- and inter-run coefficients of variation of cycle threshold of 1.1%-2.1%. The application of real-time quantitative PCR showed that T7-based RNA amplification is reproducible but might introduce a sequence-specific bias. Real-time quantitative PCR is a novel approach to monitor RNA amplification and is particularly suited to study RNA amplification of individual genes.

In this study, urine was collected from groups of volunteers following the consumption of water, ascorbic acid, or cranberry supplements. Only ascorbic acid intake consistently produced acidic urine. Photospectroscopy data indicated that increased water consumption produced urine with lower protein content. Surface tension measurements of the collected urine showed that both water and cranberry supplementation consistently produced urine with surface tensions higher than the control or urine collected following ascorbic acid intake. These urine samples were also employed to study uropathogen adhesion to silicone rubber in a parallel plate flow chamber. Urine obtained after ascorbic acid or cranberry supplementation reduced the initial deposition rates and numbers of adherent Escherichia coli and Enterococcus faecalis, but not Pseudomonas aeruginosa, Staphylococcus epidermidis, or Candida albicans. Conversely, urine obtained from subjects with increased water intake vastly increased the initial deposition rates and numbers of adherent E. coli and E. faecalis (P < 0.05).

A general method is described for using a Lisp Machine to study reading as it takes place. The method involves simulating, on the machine’s CRT, a moving window that passes through the text being read. The speed and direction of the window can be controlled by the reader, or test probes can be coordinated within the text viewed. The method allows reading to occur in an easily monitored environment, and the reader can respond to the material being read in a flexible manner. Suggestions for programming the Lisp Machine are provided which lead to improved overall execution speed and constancy of runtime for specific pieces of code in on-line experiments.