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The stereoselective synthesis of chiral molecules in enantioenriched form – i.e., asymmetric synthesis – underpins many fields of pure and applied science. Enantioconvergent reactions are preeminent in contemporary asymmetric synthesis as they convert both enantiomers of a racemic starting material into a single enantioenriched product, thus avoiding the maximum 50% yield associated with resolutions. All currently known enantioconvergent processes necessitate the loss or partial-loss of the racemic substrate’s configuration, thus limiting the potential substrate scope to molecules that contain labile stereogenic units. Here we present a new approach to enantioconvergent reactions that can proceed with full retention of the racemic substrate’s configuration. This uniquely stereo-economic approach is possible if the two enantiomers of a racemic starting material are joined together to form one enantiomer of a non-meso product. Experimental validation of this concept is presented using two distinct strategies; (1) a direct unsymmetrical coupling approach and (2) a multi-component approach, which exhibits statistical-amplification of enantiopurity. Our results demonstrate that stereoretentive-enantioconvergent reactions can be achieved using a wide variety of synthetic tactics, including stoichiometric chiral reagents, chiral auxiliaries, and chiral catalysts. Thus, the established dogma that enantioconvergent reactions require substrates that contain labile stereogenic units is shown to be incorrect. We anticipate the concept of stereoretention in enantioconvergent reactions will lead to the development of novel methodologies that can utilize a hitherto unexplored range of racemic substrates.

Supplementary Figure 1 from Phosphorylated Insulin-Like Growth Factor-I/Insulin Receptor Is Present in All Breast Cancer Subtypes and Is Related to Poor Survival

Introduction the identification and management of frailty occurs mostly in primary care. Several different models of care exist. This study aimed to assess the impact of a new General Practitioner (GP)-led modified Comprehensive Geriatric Assessment (CGA) on service delivery, healthcare utilisation and patient outcomes. Method patients with moderate–severe frailty (electronic Frailty Index score > 0.24) in Newbattle Medical Practice, Scotland, were eligible for a novel intervention (MidMed) in which an additional GP performed a modified CGA and was directly accessible for appointments. The recruits to the intervention (MidMed) group were compared with those waiting to be enrolled (non-MidMed). Outcomes included unscheduled hospital admissions, primary care consultations, continuity of care (Usual Provider of Care (UPC) index), outpatient attendances and mortality. Adjusted rate ratios (aRR), for MidMed compared to non-MidMed, were estimated using regression models adjusting for demographics and healthcare utilisation histories. Results 510 patients were included: 290 MidMed (mean(SD) age 80.1(7.6)years; 59.6% female) and 220 non-MidMed (75.4(8.6)years; 57.7% female). Median follow-up was 396 days. aRR(95%CI) was 0.46(0.30–0.71) for >1 admission, 0.62(0.41–0.95) >1 Emergency Department (ED) attendance and 1.52(1.30–1.75) for use of primary care, with no difference in outpatient appointments or mortality. Continuity of care was better for the MidMed group (MidMed UPC 0.77(SD 0.19), non-MidMed 0.41(0.18), P Conclusion this GP-led service for frail patients was associated with lower risk of hospital readmission/ED reattendance, greater use of primary care and improved continuity of care. More detailed evaluation of novel primary care frailty services, over longer time-periods, including robust randomised controlled trials, are needed.

A biomimetic synthetic strategy has resulted in a two-step total synthesis of (±)-ulodione A and the prediction of two potential natural products, (±)-ulodiones C and D. This work was guided by computational investigations into the selectivity of a proposed biosynthetic Diels-Alder dimerization, which was then utilized in the chemical synthesis. This work highlights how biosynthetic considerations can both guide the design of efficient synthetic strategies and lead to the anticipation of new natural products.

Early accurate diagnosis of infection ± organ dysfunction (sepsis) remains a major challenge in clinical practice. Utilizing effective biomarkers to identify infection and impending organ dysfunction before the onset of clinical signs and symptoms would enable earlier investigation and intervention. To our knowledge, no prior study has specifically examined the possibility of pre-symptomatic detection of sepsis.Blood samples and clinical/laboratory data were collected daily from 4385 patients undergoing elective surgery. An adjudication panel identified 154 patients with definite postoperative infection, of whom 98 developed sepsis. Transcriptomic profiling and subsequent RT-qPCR were undertaken on sequential blood samples taken postoperatively from these patients in the three days prior to the onset of symptoms. Comparison was made against postoperative day-, age-, sex- and procedure- matched patients who had an uncomplicated recovery (n =151) or postoperative inflammation without infection (n =148).Specific gene signatures optimized to predict infection or sepsis in the three days prior to clinical presentation were identified in initial discovery cohorts. Subsequent classification using machine learning with cross-validation with separate patient cohorts and their matched controls gave high Area Under the Receiver Operator Curve (AUC) values. These allowed discrimination of infection from uncomplicated recovery (AUC 0.871), infectious from non-infectious systemic inflammation (0.897), sepsis from other postoperative presentations (0.843), and sepsis from uncomplicated infection (0.703).Host biomarker signatures may be able to identify postoperative infection or sepsis up to three days in advance of clinical recognition. If validated in future studies, these signatures offer potential diagnostic utility for postoperative management of deteriorating or high-risk surgical patients and, potentially, other patient populations.

The bicyclo[2.2.2]diazaoctane alkaloids are a vast group of natural products which have been the focus of attention from the scientific community for several decades. This interest stems from their broad range of biological activities, their diverse biosynthetic origins, and their topologically complex structures, which combined make them enticing targets for chemical synthesis. In this article, full details of our synthetic studies into the chemical feasibility of a proposed network of biosynthetic pathways towards the brevianamide family of bicyclo[2.2.2]diazaoctane alkaloids are disclosed. Insights into issues of reactivity and selectivity in the biosynthesis of these structures have aided the development of a unified biomimetic synthetic strategy, which has resulted in the total synthesis of all known bicyclo[2.2.2]diazaoctane brevianamides and the anticipation of an as-yet-undiscovered congener.

The bicyclo[2.2.2]diazaoctane alkaloids are a vast group of natural products which have been the focus of attention from the scientific community for several decades. This interest stems from their broad range of biological activities, their diverse biosynthetic origins, and their topologically complex structures, which combined make them enticing targets for chemical synthesis. In this article, full details of our synthetic studies into the chemical feasibility of a proposed network of biosynthetic pathways towards the brevianamide family of bicyclo[2.2.2]diazaoctane alkaloids are disclosed. Insights into issues of reactivity and selectivity in the biosynthesis of these structures have aided the development of a unified biomimetic synthetic strategy, which has resulted in the total synthesis of all known bicyclo[2.2.2]diazaoctane brevianamides and the anticipation of an as-yet-undiscovered congener.

Background Heart rate variability (HRV) is reduced in cirrhosis and in conditions of systemic inflammation. Whether HRV is associated with cirrhosis decompensation and development of acute-on-chronic liver failure (ACLF) is unknown. Aims To (a) validate wireless remote HRV monitoring in cirrhosis decompensation; (b) determine if severely reduced HRV is a surrogate for inflammation and progression of cirrhosis decompensation; (c) assess if measuring HRV determines prognosis in cirrhosis decompensation. Methods One hundred and eleven patients at risk of cirrhosis decompensation at two clinical sites were monitored for HRV. Standard deviation of all normal beat-beat intervals (SDNN) reflecting HRV was assessed using remote monitoring (Isansys Lifetouch) and/or Holter ECG recording. Clinical outcomes and major prognostic scores were recorded during 90-day follow-up. Results Reduced HRV denoted by lower baseline SDNN, correlated with severity of decompensation (median 14 (IQR 11-23) vs 33 (25-42); P

Our study provides novel findings of experimental hypokalemia reducing urea cycle functionality and thereby severely increasing plasma ammonia. This is pathophysiologically interesting because plasma ammonia increases during hypokalemia by a hitherto unknown mechanism, which may be particular important in relation to the unexplained link between hypokalemia and hepatic encephalopathy. Potassium deficiency decreases gene expression, protein synthesis, and growth. The urea cycle maintains body nitrogen homeostasis including removal of toxic ammonia. Hyperammonemia is an obligatory trait of liver failure, increasing the risk for hepatic encephalopathy, and hypokalemia is reported to increase ammonia. We aimed to clarify the effects of experimental hypokalemia on the in vivo capacity of the urea cycle, on the genes of the enzymes involved, and on ammonia concentrations. Female Wistar rats were fed a potassium-free diet for 13 days. Half of the rats were then potassium repleted. Both groups were compared with pair- and free-fed controls. The following were measured: in vivo capacity of urea-nitrogen synthesis (CUNS); gene expression (mRNA) of urea cycle enzymes; plasma potassium, sodium, and ammonia; intracellular potassium, sodium, and magnesium in liver, kidney, and muscle tissues; and liver sodium/potassium pumps. Liver histology was assessed. The diet induced hypokalemia of 1.9 ± 0.4 mmol/L. Compared with pair-fed controls, the in vivo CUNS was reduced by 34% ( P < 0.01), gene expression of argininosuccinate synthetase 1 ( ASS1) was decreased by 33% ( P < 0.05), and plasma ammonia concentrations were eightfold elevated ( P < 0.001). Kidney and muscle tissue potassium contents were markedly decreased but unchanged in liver tissue. Protein expressions of liver sodium/potassium pumps were unchanged. Repletion of potassium reverted all the changes. Hypokalemia decreased the capacity for urea synthesis via gene effects. The intervention led to marked hyperammonemia, quantitatively explainable by the compromised urea cycle. Our findings motivate clinical studies of patients with liver disease.

Background: In cirrhosis, a pathological gut microbiome has been linked with immune dysfunction. A pilot study of probiotic Lactobacillus casei Shirota (LcS) in alcoholic cirrhosis demonstrated significant improvement in neutrophil function. This study aimed to evaluate the efficacy of LcS on neutrophil function and significant infection rates in patients with cirrhosis. Methods: 92 cirrhotic patients (Child&ndash, Pugh score &le, 10) were randomized to receive LcS or placebo, three times daily for six months. Primary end-points were incidence of significant infection and neutrophil function. Secondary end-points were cytokine profile, endotoxin, bacterial DNA positivity, intestinal permeability and quality of life. Results: Rates of infection, decompensation or neutrophil function did not differ between placebo and probiotic groups. LcS significantly reduced plasma monocyte chemotactic protein-1 and, on subgroup analysis, plasma interleukin-1&beta, (alcoholic cirrhosis), interleukin-17a and macrophage inflammatory protein-1&beta, (non-alcoholic cirrhosis), compared with placebo. No significant differences in intestinal permeability, bacterial translocation or metabolomic profile were observed. Conclusion: LcS supplementation in patients with early cirrhosis is safe. Although no significant infections were observed in either group, LcS improved cytokine profile towards an anti-inflammatory phenotype, an effect which appears to be independent of bacterial translocation.

OBJECTIVES: Caval subtraction phase-contrast magnetic resonance imaging (PCMRI) noninvasive measurements of total liver blood flow (TLBF) and hepatic arterial (HA) flow have been validated in animal models and translated into normal volunteers, but not patients. This study aims to demonstrate its use in patients with liver cirrhosis, evaluate measurement consistency, correlate measurements with portal hypertension severity, and invasively validate TLBF measurements. MATERIALS AND METHODS: Local research ethics committee approval was obtained. Twelve patients (mean, 50.8 ± 3.1 years; 10 men) with histologically confirmed cirrhosis were recruited prospectively, undergoing 2-dimensional PCMRI of the portal vein (PV) and the infrahepatic and suprahepatic inferior vena cava. Total liver blood flow and HA flow were estimated by subtracting infrahepatic from suprahepatic inferior vena cava flow and PV flow from estimated TLBF, respectively. Invasive hepatic venous pressure gradient (HVPG) and indocyanine green (ICG) clearance TLBF were measured within 7 days of PCMRI. Bland-Altman (BA) analysis of agreement, coefficients of variation, and Pearson correlation coefficients were calculated for comparisons with direct inflow PCMRI, HVPG, and ICG clearance. RESULTS: The mean difference between caval subtraction TLBF and direct inflow PCMRI was 6.3 ± 4.2 mL/min/100 g (BA 95% limits of agreement, ±28.7 mL/min/100 g). Significant positive correlations were observed between HVPG and caval subtraction HA fraction (r = 0.780, P = 0.014), but not for HA flow (r = 0.625, P = 0.053), PV flow (r = 0.244, P = 0.469), or caval subtraction TLBF (r = 0.473, P = 0.141). Caval subtraction and ICG TLBF agreement was modest (mean difference, -32.6 ± 16.6 mL/min/100 g; BA 95% limits of agreement, ±79.7 mL/min/100 g), but coefficients of variation were not different (65.7% vs 48.1%, P = 0.28). CONCLUSIONS: In this proof-of-principle study, caval subtraction PCMRI measurements are consistent with direct inflow PCMRI, correlate with portal hypertension severity, and demonstrate modest agreement with invasive TLBF measurements. Larger studies investigating the clinical role of TLBF and HA flow measurement in patients with liver disease are justified.

BACKGROUND AND AIMS: In nonalcoholic fatty liver disease (NAFLD), fibrosis is the most important factor contributing to NAFLD-associated morbidity and mortality. Prevention of progression and reduction in fibrosis are the main aims of treatment. Even in early stages of NAFLD, hepatic and systemic hyperammonemia is evident. This is due to reduced urea synthesis; and as ammonia is known to activate hepatic stellate cells, we hypothesized that ammonia may be involved in the progression of fibrosis in NAFLD.APPROACH AND RESULTS: In a high-fat, high-cholesterol diet-induced rodent model of NAFLD, we observed a progressive stepwise reduction in the expression and activity of urea cycle enzymes resulting in hyperammonemia, evidence of hepatic stellate cell activation, and progressive fibrosis. In primary, cultured hepatocytes and precision-cut liver slices we demonstrated increased gene expression of profibrogenic markers after lipid and/or ammonia exposure. Lowering of ammonia with the ammonia scavenger ornithine phenylacetate prevented hepatocyte cell death and significantly reduced the development of fibrosis both in vitro in the liver slices and in vivo in a rodent model. The prevention of fibrosis in the rodent model was associated with restoration of urea cycle enzyme activity and function, reduced hepatic ammonia, and markers of inflammation.CONCLUSIONS: The results of this study suggest that hepatic steatosis results in hyperammonemia, which is associated with progression of hepatic fibrosis. Reduction of ammonia levels prevented progression of fibrosis, providing a potential treatment for NAFLD.

Elderly patients have complex health needs and are vulnerable to inadequate or undignified treatment.1 Fostering professional and altruistic attitudes in medical education is necessary in order to prepare medical students for care of older patients.2 This paper describes a low-fidelity simulation session designed to increase empathy towards elderly patients among University of Edinburgh medical students. The session aims to enhance understanding of effects of age-related physical impairments on activities of daily living; how these impairments may challenge good clinical care and stimulate discussion about how these challenges may be mitigated. The session has been integrated into the Medicine of the Elderly teaching programme and here we describe it as delivered in NHS Fife, where the session has expanded to include a commercial age simulation suit3 to further enhance the learning experience. The 90 min session accommodates six medical students and is facilitated by two tutors in the Education Centre, Victoria Hospital, Kirkcaldy. Initially, the students are introduced to the aims during a 10 min briefing, and safety aspects are discussed. The students are divided into groups: the first focus on mobility; the second on special senses, before switching over after 30 min. Finally, there is a 20 min debrief. The students are supervised at all times. ### Mobility Each student has the opportunity to wear the simulation suit, consisting of a weighted vest, soft collar, straps and overshoes (see figure 1). The student is dressed in the components over her/his own attire by the other group members before carrying out tasks around the education centre: getting from seated to standing position; …

As postsynaptic metabotropic subtype 1 (mGlu1) receptors are present in the thalamus, we have investigated the effect of potentiating and antagonising mGlu1 receptors on responses of thalamic neurones to noxious sensory stimulation. Extracellular recordings were made in vivo with multi-barrel iontophoretic electrodes from single neurones in the thalamus of urethane-anaesthetised rats. Responses to iontophoretic applications of the Group I mGlu agonist 3,5-dihydroxy-phenylglycine (DHPG) were selectively potentiated by co-application of the mGlu1 positive allosteric modulator Ro67-4853, whereas they were selectively reduced upon co-application of the mGlu1 receptor orthosteric antagonist LY367385. This indicates that thalamic DHPG responses are mediated primarily via mGlu1 receptors, consistent with the high postsynaptic levels of this receptor in the thalamus. Furthermore, potentiation of DHPG responses by Ro67-4853 were greater when the initial DHPG response was of a low magnitude. Ro67-4853 also potentiated responses of thalamic neurones to noxious thermal stimulation, whilst having little effect on the baseline activity of nociceptive neurones. By contrast, nociceptive responses were reduced by LY367385. In a further series of experiments we found that inactivation of somatosensory cortex by cooling resulted in a reduction of thalamic nociceptive responses. These results underline the importance of mGlu1 receptors in the processing of sensory information in the thalamus, particularly with respect to nociceptive responses. Furthermore, the involvement of mGlu1 receptors may reflect the activity of descending cortico-thalamic afferents., Highlights • Pharmacological potentiation of mGlu1 receptors enhances nociceptive responses of thalamic neurones. • Antagonism of mGlu1 receptors reduces nociceptive responses of thalamic neurones. • Inactivation of somatosensory cortex reduces nociceptive responses of thalamic neurones. • This suggests a role for mGlu1 receptors and cortico-thalamic pathways in nociceptive processing.

Graphical abstract, Highlights • We investigated the influence of area MT feedback on LGN cell responses to visual stimuli. • We used focal GABA micro-iontophoresis to reversibly block area MT cell responses. • Inactivating area MT feedback produced clear and reversible changes in LGN cell responses. • Effects were observed across magno, parvo and koniocellular LGN cell types., The feedback connections from the cortical middle temporal (MT) motion area, to layer 6 of the primary visual cortex (V1), have the capacity to drive a cascaded feedback influence from the layer 6 cortico-geniculate cells back to the lateral geniculate nucleus (LGN) relay cells. This introduces the possibility of a re-entrant motion signal affecting the relay of the retinal input through the LGN to the visual cortex. The question is whether the response of LGN cells to moving stimuli involves a component derived from this feedback. By producing a reversible focal pharmacological block of the activity of an MT direction column we show the presence of such an influence from MT on the responses of magno, parvo and koniocellular cells in the macaque LGN. The pattern of effect in the LGN reflects the direction bias of the MT location inactivated. This suggests a moving stimulus is captured by iterative interactions in the circuit formed by visual cortical areas and visual thalamus.

The projections between the thalamus and primary visual cortex (V1) are a key reciprocal neural circuit, relaying retinal signals to cortical layers 4 & 6 while being simultaneously regulated by massive layer 6 corticothalamic feedback. Effectively dissecting the influence of this corticothalamic feedback circuit in higher mammals remains a challenge for vision research. By pharmacologically increasing the focal gain of visually driven layer 6 responses of cat V1 in a controlled fashion, we examined the effects of such focal cortical changes on the response amplitudes and spatial structure of the receptive fields (RFs) of individual dorsal lateral geniculate nucleus (dLGN) cells. We found that enhancing visually driven cortical feedback could facilitate or suppress the overall responses of dLGN cells, and such an effect was linked to the orientation preference of the cortical neuron. Related to these selective retinotopic gain changes, enhanced feedback induced the RFs of dLGN cells to expand, contract or shift their spatial focus. Our results provide further evidence for a functional mechanism through which the cortex can selectively gate visual information flow from the thalamus back to the visual cortex.

Background: selective serotonin reuptake inhibitors (SSRIs) may affect the neurodegenerative process of dementia, enhancing cognition. This systematic review aims to determine whether SSRIs influence cognitive performance, mood and function in people with any type of dementia.Method: randomised placebo-controlled studies of SSRIs in people with dementia, which recorded cognitive outcomes, were identified in ALOIS (ALzheimer's and cOgnitive Improvement Studies register) in April 2013 and updated in January 2015. Data were extracted on cognition, agitation, mood, activities of daily living (ADLs) and adverse events. End of treatment statistics were calculated.Results: twelve studies met inclusion criteria (1,174 participants), of which seven studies (710 participants) provided data for meta-analysis on cognition. There was no difference in MMSE score at end of treatment; mean difference (MD) was 0.28 (95% CI −0.83 to 1.39) (six studies, 470 participants). For change in MMSE scores, there was a small improvement; MD was 0.53 (95%CI −0.07 to 1.14) (three studies, 352 participants). The remaining studies showed no improvement in cognition. There was no statistically significant benefit of SSRIs on mood (four studies, 317 participants); standard mean difference (SMD) −0.10 (95% CI −0.39 to 0.2), agitation (three studies, 189 participants); SMD −0.01(95% CI −0.86 to 0.83), or ADLs at end of treatment (four studies, 336 participants); SMD −0.15(95% CI −0.45 to 0.15). There was no difference in mortality between the two groups. Study quality was mixed with concerns over incomplete data.Conclusion: a small number of relatively low-powered studies showed no benefit or harm from SSRIs in terms of cognition, mood, agitation or ADLs. Large, methodologically robust studies are needed.

Metabotropic glutamate subtype 1 (mGlu1) receptor is thought to play a role in synaptic responses in thalamic relay nuclei. The aim of this study was to evaluate the positive allosteric modulator (PAM) Ro67-4853 as a tool to modulate thalamic mGlu1 receptors on single thalamic neurones in vivo. Ro67-4853, applied by iontophoresis onto ventrobasal thalamus neurones of urethane-anaesthetised rats, selectively enhanced responses to the agonist (S)-3,5-dihydroxy-phenylglycine (DHPG), an effect consistent with mGlu1 potentiation. The PAM was also able to enhance maintained responses to 10 Hz trains of sensory stimulation of the vibrissae, but had little effect on responses to single sensory stimuli. Thus Ro67-4853 appears to be a highly selective tool that can be useful in investigating how mGlu1 receptor potentiation can alter neural processing in vivo. Our results show the importance of mGlu1 in sensory processing and attention mechanisms at the thalamic level and suggest that positive modulation of mGlu1 receptors might be a useful mechanism for enhancing cognitive and attentional processes., Highlights ► We the studied effects of Ro67-4853, an mGlu1 PAM, on single thalamic cells in vivo. ► Ro67-4853 selectively potentiated responses to the agonist DHPG. ► Ro67-4853 potentiated sensory synaptic responses to repetitive vibrissa stimulation. ► Ro67-4853 is a selective tool to potentiate mGlu1-mediated neural processing in vivo. ► Positive modulation of mGlu1 may be a useful means to enhance cognition and attention.

To test if an arbitrary definition of day and night periods that differs to patient-reported awake and sleep periods leads to inaccuracies in interpretation of ambulatory blood pressure monitoring (ABPM). A single-center, retrospective review was performed comparing three different methods to classify day and night periods following a single 24-h ABPM: method A: Patient's record during monitoring; method B: 7:00 am to 11:00 pm day and 11:00 pm to 7:00 am night; method C: 8:00 am to 8:00 pm day and midnight to 6:00 am night. We included 149 studies in 149 children with a mean ± SD age of 13.0 ± 3.4 years. Reported sleep duration was 9.2 ± 1.3 h. Significant differences resulted between three methods for the means of several ambulatory BP parameters including indexed BP values during day, BP load, and nocturnal dipping status. During monitoring [median (range)], 7.5% (0.0-27.5%) readings were misclassified using method B and 0.0% (0.0-20.0%) using method C (p 0.0001). This misclassification resulted in change of hypertension status for 11.4% (17/149) patients using method B and 9.4% (14/149) patients using method C (p = 0.70). Misclassification of measurements during ABPM can introduce significant errors in its interpretation. The clinical impact of these findings needs further evaluation in larger prospective studies.

PURPOSE To evaluate a credit portfolio process for accessibility, relevance, validity and sustainability, and provide evidence for credit requirements for certification maintenance. METHODS Certified diabetes educators (CDEs) who had recertified by examination from 2001 to 2003 were invited to participate. CDEs completed a satisfaction survey and recorded retrospectively credited learning activities over 2 years. Participants were asked to describe the learning activity, identify the learning category and credits allocated, relate the activity to CDE competencies, review the activity definition/documentation requirements and make suggestions for improvement. RESULTS Seventy-four of 266 CDEs responded (28%). CDEs engaged in an average of 92 (± 40) credits of professional development activities per year. Registered nurses (RNs) and registered dietitians (RDs) participated equally in a wide variety of activities. Nurses earned a significantly greater number of credits: 224.8 for RNs and 157.4 for RDs (p CONCLUSIONS CDEs engage in a wide variety of professional development activities and can provide evidence of this when requested. This study supports a requirement of 250 credits over 5 years for certification maintenance.

Growth factors provide powerful mitogenic and survival signals to breast cancer cells and it is therefore not surprising that they are able to subvert inhibitory responses to anti-hormonal drugs. In this review we discuss several mechanisms by which this may be achieved and expand our observations to encompass recently emerging anti-growth factor treatments. The information presented is underpinned by inhibitor studies that show the targeting of such mechanisms in advance of anti-hormone or anti-growth factor resistance development is able to substantially delay this event, thus pointing the way forward to intelligent combination therapies relevant to the future management of breast cancer.

Resistance to antiepidermal growth factor (EGFR) strategies is an emerging clinical problem. Using human colorectal cancer (CRC) cells, we evaluated the involvement of the insulin receptor isoform-A (InsR-A) in de novo resistance to gefitinib, an EGFR tyrosine kinase inhibitor. Challenging the EGFR positive LoVo cells with gefitinib (1 microM) resulted in a small ( approximately 18%) inhibition of cell growth and although a modest reduction in phospho (p)EGFR Tyr845 was seen, pEGFR at residues -Tyr1068 and -Tyr1173 were unchanged. LoVo cells produced unprocessed pro-IGF-1R protein, substantial levels of IGF-II mRNA and mature InsR protein, consisting mainly of the InsR-A isoform. Insulin and IGF-II promoted cell growth and pEGFR Tyr845, Tyr1068 and Tyr1173 activity and conversely, the insulin-like growth factor-1 receptor (IGF-1R)/InsR inhibitor ABDP (1 muM) inhibited growth and reduced pEGFR activity at all three tyrosine residues. pInsR and pAkt levels were increased after gefitinib treatment. Blocking of pInsR with ABDP enabled gefitinib to markedly reduce pEGFR Tyr845, Tyr1068 and Tyr1173. Short-term gefitinib/ABDP dual treatment was more effective than either agent alone and chronic exposure to this combination resulted in total cell loss after 9 weeks, preventing acquisition of resistance to ABDP. LoVo cells with acquired resistance to ABDP were acutely sensitive to gefitinib. We concluded that InsR-A reduces sensitivity to gefitinib in LoVo CRC cells, thus its co-targeting alongside EGFR can improve the anti-tumour effect of gefitinib.

Over-expression or aberrant signalling of the erbB family members epidermal growth factor receptor (EGFR) and HER2 (erbB2/neu) have been associated with the pathogenesis of the malignant phenotype. In addition, high levels of EGFR and HER2 expression have been shown to correlate with poor prognosis and also implicated in disease progression. Signal transduction inhibitors (STIs) have been developed with specifically target these receptors and include the small molecule tyrosine kinase inhibitor gefitinib (IressaTM) which targets the EGFR and the humanised monoclonal antibody trastuzumab (HerceptinTM), which has anti-tumour activity against HER2. Studies however, have indicated that de novo or acquired resistance to these agents is a major clinical problem. Cancer cells are highly adaptive and can readily switch from one receptor signalling pathway to another in order to maintain growth or cell survival, a process paradoxically, that in many instances is induced by the anti-tumour agents themselves, ultimately limiting their activity and promoting resistance. Evidence is accumulating which demonstrates that signalling interplay occurs between the EGFR/HER2 and the insulin-like growth factor -1 receptor (IGF-1R) and the article will focus on the growth factor pathway switching that occurs between these receptors which can influence the effectiveness gefitinib and trastuzumab.

Insulin-like growth factor-I receptor (IGF-IR) signaling is involved in many fundamental adverse aspects of cancer cell biology, such as proliferation, cell survival and migration. Its anti-apoptotic properties have implicated the receptor in mediating decreased sensitivity to chemotherapeutic drugs and radiation treatment; however, data are emerging that also indicates a role for IGF-IR signaling in resistance, not only to antihormones but also to antigrowth factor strategies such as agents that target the erb family of receptors. As such, IGF-IR is clearly an attractive therapeutic target for the treatment of cancer, including breast cancer, where there is evidence of clinical prominence of the IGF-IR pathway and, as such, numerous strategies are currently in development to inhibit IGF-IR signaling. This review focuses on the ability of the IGF-IR to contribute to resistance mechanisms that support breast cancer cell growth in the presence of antihormones and antigrowth factors and discusses methods to maximize antitumor effects by combination regimens cotargeting the IGF-IR that may delay, or even prevent, progression to the resistant phenotype.

Ring diameters and ring number counts were recorded from Calluna stem cross-sections harvested during an N manipulation experiment. The application of dendrochronological methods permitted accurate ageing of shrubs growing in a heathland of known age. Linear regression analyses demonstrated a highly significant association between increasing levels of N input and stem diameter in both watered and droughted plots (droughted in 1997 only). N inputs were identified as the most significant environmental stressor, appearing to speed ageing vis the Calluna growth cycle. Drought sensitivity was noted in the high N treatment plots (120 kg N ha−1 yr−1) with decreased stem diameters. This may have implications in the response of Calluna growth under current climate change scenarios.

Previous studies on the effects of herbivores on nutrient cycling have given little consideration to the relationship between soil nitrogen (N) and phosphorus (P) availability. Here we examined how browsing by red deer influences the relative availability of N and P in a regenerating woodland ecosystem. We found that removal of browsing by fencing for 14 years led to a shift from N toward P limitation of the dominant tree species Betula pubescens. This was evidenced by a significant increase in foliar N:P ratio of B. pubescens as a result of removal of browsing; mean N:P ratio of foliage from browsed areas was 13.2 suggesting that trees growing in browsed areas were N limited, whereas foliage from unbrowsed areas had a mean N:P ratio of 15.8, suggesting that these areas were more P limited. Further evidence of a shift toward P limitation in unbrowsed areas came from the finding that root uptake of labelled 32P was significantly greater in roots collected from unbrowsed than browsed trees. Soil phosphatase activity did not significantly differ between browsed and unbrowsed areas. Our data indicate therefore that herbivores have the potential to significantly affect the stoichiometry of N and P in forest ecosystems.

Anti-hormones (notably tamoxifen), chemotherapy and modern radiotherapeutic approaches are invaluable in the management of breast cancer, and collectively have contributed substantially to the improved survival in this disease. Moreover, there is promise that these successes will continue with the emergence of other endocrine agents (for example, aromatase inhibitors and pure anti-oestrogens). However, de novo and acquired resistance comprises a significant problem with all treatment approaches examined to date. This Workshop aimed to evaluate the contribution made by growth factor signalling pathways in the various resistant states, primarily focusing on resistance to anti-hormonal strategies and spanning experimental models and, where possible, clinical breast cancer data. The successes and limitations of therapeutic targeting of these pathways with various signal transduction inhibitors (STIs) were evaluated in model systems and from emerging clinical trials (including epidermal growth factor receptor inhibitors such as gefitinib). It was concluded that growth factor signalling is an important contributor in the development of endocrine resistance in breast cancer and that use of STIs provides a promising therapeutic strategy for this disease. However, the cancer cell is clearly able to harness alternative growth factor signalling pathways for growth and cell survival in the presence of STI monotherapy and, as a consequence, the efficacy of STIs is likely to be limited by the acquisition of resistance. A number of strategies were proposed from studies in model systems that appeared to enhance anti-tumour actions of existing STI monotherapy, notably including combination therapies targeting multiple pathways. With the increased availability of diverse STIs and improved drug delivery, there is much hope that the more complex therapeutic strategies proposed may ultimately be achievable in clinical practice.

Apoptotic nerve cell death is implicated in the pathogenesis of several devastating neurodegenerative conditions, including glaucoma and Alzheimer's and Parkinson's diseases. We have devised a noninvasive real-time imaging technique using confocal laser-scanning ophthalmoscopy to visualize single nerve cell apoptosis in vivo , which allows longitudinal study of disease processes that has not previously been possible. Our method utilizes the unique optical properties of the eye, which allow direct microscopic observation of nerve cells in the retina. We have been able to image changes occurring in nerve cell apoptosis over hours, days, and months and show that effects depend on the magnitude of the initial apoptotic inducer in several models of neurodegenerative disease in rat and primate. This technology enables the direct observation of single nerve cell apoptosis in experimental neurodegeneration, providing the opportunity for detailed investigation of fundamental disease mechanisms and the evaluation of interventions with potential clinical applications, together with the possibility of taking this method through to patients.

An investigation was conducted to determine the impact of acute doses of gamma radiation on the microbial community structure of a Holcus lanatus dominated grassland soil. Mesocosms containing soil and established grass were irradiated using a sealed 137Cs source (7.0 Gy min–1). Doses ranged from 5 to 160 Gy, analyses were conducted on the day of irradiation, then 7 and 30 days later. Plant growth and arbuscular mycorrhizal fungal colonisation of roots were reduced by irradiation. Gram-negative bacteria, and microbial metabolic capacity were also negatively affected by treatment. Microbial biomass measured by phospholipid fatty acid (PLFA) analysis, showed an increase at doses above 20 Gy, 7 and 30 days after treatment. Proportions of Gram-positive bacterial and fungal PLFAs fluctuated inversely to each other, in response to both sampling time and radiation dose. We hypothesise that many of the observed soil microbial responses are indirect effects mediated by the influence of ionising radiation on the plants in this system.

An increasing body of evidence demonstrates that growth factor networks are highly interactive with estrogen receptor signaling in the control of breast cancer growth. As such, tumor responses to antiestrogens are likely to be a composite of the estrogen receptor and growth factor-inhibitory activity of these agents, with alterations/aberrations in growth factor signaling providing a mechanism for the development of antiestrogen resistance. In this light, the current article focuses on illustrating the relationship between growth factor signaling and antiestrogen failure in our in-house tumor models of breast cancer and describing how we are now beginning to successfully target growth factor activity to improve the effects of antiestrogen drugs and to block aggressive disease progression.

The diagnosis of tree nutrient demand for nitrogen (N), phosphorus (P), and potassium (K) is possible using a root bioassay with excised roots, to measure the rate of uptake of the corresponding isotope. In this study, Norway spruce (Picea abies) roots were used to test the uptake response in roots of different diameter. The 15 N bioassay has been developed to test for nitrogen demand by plants, where high 15 N uptake demonstrates nitrogen limitation. Sometimes sample variability may mask potential treatment differences, but we have shown that when the fine root fraction of

The development of acquired resistance to antihormonal agents in breast cancer is a major therapeutic problem. We have developed a tamoxifen-resistant (TAM-R) MCF-7 breast cancer cell line to investigate the mechanisms behind this condition. Both epidermal growth factor receptor (EGFR) and c-erbB2 mRNA and protein expression were increased in TAM-R compared with wild-type MCF-7 cells, whereas comparable levels of c-erbB3 mRNA and protein were expressed in both cell lines. Under basal conditions, phosphorylated EGFR/c-erbB2, EGFR/c-erbB3 but not c-erbB2/c-erbB3 receptor heterodimers were detected in TAM-R cells in association with increased levels of phosphorylated extracellular-signal regulated kinase 1/2 (ERK1/2). Both cell lines were capable of generating a range of EGFR-specific ligands and increased expression of transforming growth factor α was observed in TAM-R cells. Treatment of TAM-R cells with ZD1839 (Iressa) or trastuzumab (Herceptin) blocked c-erbB receptor heterodimer formation and phosphorylation, reduced ERK1/2 activity, and strongly inhibited cell growth. The MAPK kinase inhibitor PD098059 specifically reduced phosphorylated ERK1/2 levels and inhibited TAM-R growth. All three agents abolished ERK1/2 activity in wild-type cells but caused only small reductions in cell proliferation. These results demonstrate that TAM-R MCF-7 cell growth is mediated by the autocrine release and action of an EGFR-specific ligand inducing preferential EGFR/c-erbB2 dimerization and downstream activation of the ERK pathway.

[Abstract] Figure-ground discrimination refers to the perception of an object, the figure, against a nondescript background. Neural mechanisms of figure-ground detection have been associated with feedback interactions between higher centers and primary visual cortex and have been held to index the effect of global analysis on local feature encoding. Here, in recordings from visual thalamus of alert primates, we demonstrate a robust enhancement of neuronal firing when the figure, as opposed to the ground, component of a motion-defined figure-ground stimulus is located over the receptive field. In this paradigm, visual stimulation of the receptive field and its near environs is identical across both conditions, suggesting the response enhancement reflects higher integrative mechanisms. It thus appears that cortical activity generating the higher-order percept of the figure is simultaneously reentered into the lowest level that is anatomically possible (the thalamus), so that the signature of the evolving representation of the figure is imprinted on the input driving it in an iterative process. Biotechnology and Biological Sciences Research Council (United Kingdom); G022305/1 Medical Research Council, (United Kingdom); G0701535

Background. The hepatitis B (HB) vaccination regime currently recommended for use in the UK for both preventative and post-exposure purposes is the accelerated regime, although there have been no recent reports of its efficacy. This observational study reports on the response rate achieved and longevity of protection conferred with this regime in a large number of haemodialysis patients following an episode of HB exposure. Methods. One-hundred and five patients received primary vaccination (vaccine administered at 0, 1 and 2 months). Eighty-six completed the regime, receiving a booster dose at month 12. Measuring antibodies to HB surface antigen (anti-HBS) 6 weeks after receiving the third and fourth doses assessed patients’ response. Seventy-seven patients subsequently had anti-HBs measured at month 24. Results. The response rate (anti-HBS )10 mIUuml) to primary vaccination and the complete regime was 33 and 73%, respectively. Non-European patients responded better to primary vaccination than Europeans (Ps0.014). Those receiving steroids responded less well to the complete vaccination regime (Ps0.007). Patient’s age, sex, renal diagnosis, diabetes mellitus, time on dialysis, dialysis adequacy, erythropoietin dose, hepatitis C or body weight did not affect response rates. By month 24, 24 responders (44%) had lost seroprotection. Antibody levels achieved with vaccination by transient responders was significantly lower than persistent responders. No patients became HB surface antigen positive during the 2-year study. Conclusion. Reserving the accelerated vaccination to the post-exposure scenario will expose many more patients to the risk of HB cross-infection than if used prophylactically. Regular monitoring is required if seroprotection is to be maintained.

As in eukaryotes, bacterial free Ca(2+) can play an important role as an intracellular signal. However, because free Ca(2+) is difficult to measure in live bacteria, most of the evidence for such a role is indirect. Gram-negative bacteria also have an outer membrane separating the external fluid from the periplasm as well as the cytosol where most bacterial metabolism takes place. Here we report, for the first time, direct measurement of free Ca(2+) in the periplasmic space of living Escherichia coli. Periplasmic free Ca(2+) was measured by targeting the Ca(2+)-activated photoprotein aequorin to this compartment using the N-terminal OmpT signal sequence. Cytosolic free Ca(2+) was determined using aequorin alone. We show that, under certain conditions, the periplasm can concentrate free Ca(2+), resulting in the inner membrane being exposed to free Ca(2+) concentrations several fold higher than in the bulk external fluid. Manipulation of periplasmic membrane-derived oligosaccharides (MDOs) altered the free Ca(2+) as predicted by the Donnan potential. With micromolar concentrations of external free Ca(2+), the periplasm concentrated free Ca (2+) some three to sixfold with respect to the external medium. A Ca(2+) gradient also existed between the periplasm and the cytosol under these conditions, the periplasmic free Ca(2+) being some one to threefold higher. At millimolar levels of external free Ca(2+), a similar concentration was detected in the periplasm, but the bacteria still maintained tight control of cytosolic free Ca(2+) in the micromolar range. We propose that the highly anionic MDOs in the periplasmic space generate a Donnan potential, capable of concentrating Ca(2+) in this compartment, where it may constitute a sink for regulation of Ca(2+)-dependent processes in the cytoplasm.

Induction of PG G/H synthase-2 (PGHS-2), a key rate-limiting enzyme in the PG biosynthetic pathway, has been implicated in prostatic adenocarcinomas in humans and dogs in vivo, but the molecular control of PGHS-2 expression in prostate cancer remains poorly understood. Using the dog model, the specific objectives of this study were to clone and characterize canine PGHS-2, and to study the regulation of its transcript, protein, and activity in a canine prostatic adenocarcinoma (CPA) cell line in vitro. The canine PGHS-2 cDNA was cloned by a combination of cDNA library screening and 5'-rapid amplification of cDNA ends, and shown to contain a 5'-untranslated region of 28 bp, an open reading frame of 1815 bp, and a 3'-untranslated region of 1655 bp. The open reading frame encodes a 604-amino acid protein that is 89% identical to the human homolog. The regulation of PGHS-2 protein and PGE(2) synthesis was studied in CPA cells cultured in the absence or presence of graded doses of phorbol 12-myristate 13-acetate (PMA), TNFalpha, and lipopolysaccharides. Results from immunoblots, immunocytochemistry, and RIAs showed that PGHS-2 protein and PGE(2) were present at low levels in control cells and were significantly induced after agonist treatment (P0.05), with PMA being the strongest inducer. Northern blot analyses also revealed a significant increase of PGHS-2 mRNA by PMA, TNFalpha, and lipopolysaccharides treatment (P0.05). Agonist-dependent induction of PGHS-2 mRNA was not dependent on new protein synthesis (coincubation with cycloheximide; 10 microg/ml) but was blocked by transcription inhibitor actinomycin D (5 microg/ml), suggesting that PGHS-2 acts an immediate early-response gene in prostatic epithelial cells. Thus, this study characterizes for the first time the structure of canine PGHS-2 and provides an in vitro model to unravel the molecular basis of PGHS-2 expression in prostatic adenocarcinomas.

We investigated the spatial organization of surround suppression in primate primary visual cortex (V1). We utilized drifting stimuli, configured to extend either from within the classical receptive field (CRF) to surrounding visual space, or from surrounding visual space into the CRF or subdivided to generate direction contrast, to make a detailed examination of the strength, spatial organization, direction dependence, mechanisms, and laminar distribution of surround suppression. Most cells (99/105, 94%) through all cortical layers, exhibited suppression (mean reduction 67%) to uniform stimuli exceeding the CRF, and 43% exhibited a more than 70% reduction. Testing with an annulus revealed two different patterns of surround influence. Some cells (37% of cells), classical surround suppression (CSS) cells exhibited responses to an annulus encroaching on the CRF that were less than the plateau in the spatial summation curve. The majority (63%), center-gated surround suppression (CGSS) cells, showed responses to annuli that equaled or exceeded the plateau in the spatial summation curve. Analysis suggested the CSS mechanism was implemented in all cells while the CGSS mechanism was implemented in varying strength across the sample with the extreme reflected in cells that gave larger responses to annuli than to a center stimulus. Reversing the direction of motion of the portion of the stimulus surrounding the CRF revealed four different patterns of effect: no reduction in the degree of suppression (22% of cells), a reduction in surround suppression (41%), a facilitation of the response above the level to the inner stimulus alone (37%), and a facilitation of the response above that to the inner stimulus alone that also exceeded the values associated with an optimal inner stimulus. The facilitatory effects were only seen for reverse direction interfaces between the central and surrounding stimulus at diameters equal to or more than the CRF size. The zones driving the suppressive influences and the direction contrast facilitation were often spatially heterogeneous and for a number of cells bore strong comparison with the class of behavior reported for surround mechanisms in MT. This suggests a potential role, for example, in extracting information about motion contrast in the representation of the three dimensional structure of moving objects.

We screened a cDNA library generated from harvested and stored sporophores of Agaricus bisporus and identified 19 genes with higher transcript levels than at the time of harvest. Five of these genes had no detectable mRNA levels prior to detachment from the mycelium. Sequence analysis of ten clones revealed significant similarities to known genes, these code for proteins involved in polymer breakdown and metabolism, cell wall synthesis, stress tolerance, cytochrome P450 activity and DNA binding. The diversity of functions of these genes suggests the changes in the sporophore after harvest involve several different physiological processes.

There is an increasing body of evidence demonstrating that growth factor networks are highly interactive with oestrogen receptor (ER) signalling in the control of breast cancer growth. As such, tumour responses to anti- hormones are likely to be a composite of the ER and growth factor inhibitory activity of these agents. The current article examines the modulation of growth factor networks during endocrine response, and presents in vitro and clinical evidence that epidermal growth factor receptor signalling, maintained in either an ER-dependent or -independent manner, is critical to anti- hormonal-resistant breast cancer cell growth. The considerable potential of the epidermal growth factor receptor-selective tyrosine kinase inhibitor, ZD 1839 (Iressa; AstraZeneca) to efficiently treat, and perhaps even prevent, endocrine-resistant breast cancer is highlighted.

Summary • The patterns of nitrogen (N) utilization and of N isotope fractionation were determined when two ecto-(ECM) and an ericoid (ERM) mycorrhizal fungus were grown with inorganic (ammonium or nitrate) or organic (glutamic acid or glycine) N sources of predetermined N isotope composition. • All N sources were readily utilized by each of the fungi but substantial differences in the pattern of N isotope fractionation were observed both between the fungi and the N sources. • Whereas several of the ECM-N source combinations exhibited significant net fractionation in favour of 15N, no such effect was seen in the ERM fungus, where, on ammonium, there was preferential assimilation of 14N. • It is concluded that isotopic fractionation during N uptake and metabolism can cause significant shifts in the 15N abundance of mycorrhizal fungi and that, as a result, any attempt to use the tissue 15N abundance as a means of identifying the substrates being exploited by mycorrhizal fungi, or their plant partners, in nature, are likely to be unrealistic.

Summary • Discrimination between 14N and 15N isotopes during assimilation of nitrogen (N) from inorganic (NO3, NH4) and organic (glutamic acid, glycine) sources by subarctic ecotypes of Eriophorum vaginatum, Luzula wahlenbergii. Betula nana and Vaccinium vitis-idaea was studied under axenic laboratory conditions. • Ectomycorrhizal B. nana and ericoid mycorrhizal V. vitis-idaea assimilated significant amounts of all the N sources, whereas nonmycorrhizal E. vaginatum and L. wahlenbergii used mineral N but had very limited abilities to use amino acids. • Root, shoot and whole-plant 15N abundance frequently differed from the sources and were influenced by mycorrhizas, indicating that plant 15N abundance is not a reliable guide to that of the N source used. • This study establishes that differences in leaf 15N abundance between coexisting plants may result from species-specific differences in isotopic fractionation of N. This challenges the view that 15N abundance in shoots of different plant functional types directly reflects their utilization of isotopically distinct soil N pools.

Streptococcus pneumoniae est une bacterie pathogene extracellulaire, transformable par l’ADN en solution. Il a ete montre que le transport du calcium joue un role cle dans sa croissance vegetative, la competence pour la transformabilite genetique et la virulence experimentale. Afin de preciser la localisation du calcium dans la bacterie, nous avons clone l’ADN complementaire de l’apoaequorine dans le chromosome de Streptococcus pneumoniae. Ceci a permis la reconstitution du systeme aequorine in vivo et les mesures par chimioluminescence de la concentration cytoplasmique en calcium libre. La concentration cytoplasmique en calcium est de 2 µM a l’equilibre et peut atteindre 14 µM apres addition de calcium dans la suspension bacterienne. Cette augmentation de la concentration intracytoplasmique en calcium libre en reponse a un gradient exterieur de calcium implique un transport actif sensible au DMB et depend de la vitesse initiale du transport de calcium.