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Markus Joerger,1 Kira-Lee Koster,1 Tomas Janik,2 Floris A de Jong3,4 1Department of Medical Oncology and Hematology, Cantonal Hospital St. Gallen, St. Gallen, Switzerland; 2Research & Development Department, Mundipharma Research Limited, Cambridge, UK; 3Global Medical Affairs Department, Mundipharma Research Limited, Cambridge, UK; 4Medical Affairs Department, Exact Sciences International GmbH, Baar, SwitzerlandCorrespondence: Tomas Janik, Mundipharma Research Limited, Cambridge Science Park, Milton Road, Cambridge, UK, Email tomas.janik@mundipharma.czPurpose: Immune checkpoint inhibitors (CPIs) have been widely adopted in a number of early and advanced malignancies. Histone deacetylase inhibitors (HDACis) and alkylating agents (AAs) have been suggested to potentiate the actions of CPIs on tumor cells. We conducted a comprehensive literature review to explore the potential synergistic activity between CPIs, AAs, and HDACis.Patients and Methods: Clinical and non-clinical studies describing outcomes in patients with cancer receiving CPIs and either concomitant or sequential (pre- or post-CPI) AAs or HDACis were identified in PubMed using pre-defined search strings. Manual searches of key oncology congresses were similarly performed. All relevant articles and abstracts were manually screened for relevance, classified according to the specific anticancer agents used (CPIs, AAs, or HDACis), tumor entity, and whether treatment was concomitant or sequential.Results: Overall, 227 unique clinical studies across a range of tumor types, both solid tumors and hematological malignancies, were identified. One hundred and fifty-nine publications on Phase I and II clinical studies together with 41 publications on Phase III studies were examined. The most commonly investigated tumor types were melanoma, triple-negative breast cancer, non-small cell lung cancer, and Hodgkin lymphoma. The randomized clinical studies identified, all of which reported on the combination of a CPI with an AA, demonstrated superior outcomes in the combination arm compared with CPI or AA monotherapy. Similarly, combination therapy with CPIs and HDACis demonstrated promising activity.Conclusion: Sequential or concomitant administration of a CPI with an AA or an HDACi may improve outcomes for patients with a range of tumor types. There is a rationale to support further investigation into the potential for synergy between CPIs, alkylating agents and/or HDACis in both the non-clinical and clinical settings.Plain Language Summary: People being treated for cancer will often receive more than one drug at a time, and the concept of combining cancer drugs is frequently investigated as a potential opportunity to improve outcomes for patients. We reviewed the published literature for clinical trials and work undertaken in laboratories to explore whether combining targeted agents that stop cancer cells from multiplying (known as checkpoint inhibitors) with traditional chemotherapy that kills cancer cells could be a useful approach. We looked at evidence in publications where checkpoint inhibitors were used at the same time as chemotherapy, or given immediately before or after chemotherapy. The most important evidence came from clinical trials where outcomes for patients receiving combinations of treatment were directly compared with those from patients receiving a single treatment. These studies showed superior outcomes for patients who were treated with a combination of cancer drugs compared with patients receiving monotherapy. We also found evidence that adding another class of cancer drug, called histone deacetylase inhibitors, might sensitize tumors to checkpoint inhibitors. These findings provide a rationale for examining alkylating agents and/or histone deacetylase inhibitors combined with checkpoint inhibitors.Keywords: histone deacetylase inhibitor, checkpoint inhibitor, alkylating agents, synergy, hematological malignancies, solid tumors

We present the case of a 70-year-old never-smoking female patient with epidermal growth factor receptor ( EGFR ) p.L858R-mutated metastatic non-small cell lung cancer (NSCLC). After three months of first-line treatment with erlotinib, progression occurred and platinum/pemetrexed was initiated, followed by a response for more than two years. After the progression, the molecular testing of a vertebral metastasis revealed a ROS proto-oncogene 1 ( ROS1 ) translocation and a human epidermal growth factor receptor 2 ( HER 2) p.S310F mutation, in addition to the known EGFR p.L858R mutation. Crizotinib then led to a durable response of 17 months. The molecular retesting of the tumour cells obtained from the recurrent pleural effusion revealed the absence of the ROS1 translocation, whereas the EGFR and HER2 mutations were still present. Afatinib was added to the crizotinib, and the combination treatment resulted in another durable response of more than two years. The patient died more than 7 years after the initial diagnosis of metastatic NSCLC. This case demonstrates that the repeated molecular testing of metastatic NSCLC may identify new druggable genomic alterations that can impact the patient management and improve the patient outcome.

Background: Immune checkpoint inhibitors (ICIs) improve overall survival (OS) in advanced/metastatic urothelial cancer (a/mUC) patients. Preliminary evidence suggests a prognostic role of inflammatory biomarkers in this setting. We aimed to develop a disease-specific prognostic inflammatory index for a/mUC patients on ICIs., Methods: Fifteen variables were retrospectively correlated with OS and progression-free survival (PFS) in a development (D, n = 264) and a validation (V, n = 132) cohort of platinum-pretreated a/mUC pts receiving ICIs at L2 or further line. A nomogram and inflammatory prognostic index (U-IPI) were developed. The index was also tested in a control cohort of patients treated with chemotherapy only (C, n = 114)., Results: The strongest predictors of OS were baseline platelet/lymphocyte (PLR) and neutrophil/lymphocyte (NLR) ratios, and lactate dehydrogenase (LDH), NLR, and albumin changes at 4 weeks. These were used to build the U-IPI, which can distinctly classify patients into good or poor response groups. The nomogram scoring is significant for PFS and OS ( p < 0.001 in the D, V, and combined cohorts) for the immunotherapy (IO) cohort, but not for the control cohort., Conclusions: The lack of a baseline systemic inflammatory profile and the absence of early serum inflammatory biomarker changes are associated with significantly better outcomes on ICIs in a/mUC pts. The U-IPI is an easily applicable dynamic prognostic tool for PFS and OS, allowing for the early identification of a sub-group with dismal outcomes that would not benefit from ICIs, while distinguishing another that draws an important benefit.

Background: TLD-1 is a novel liposomal doxorubicin that compared favorably to conventional doxorubicin liposomal formulations in preclinical models. This phase I first-in-human study aimed to define the maximum tolerated dose (MTD), recommended phase 2 dose (RP2D), safety and preliminary activity of TLD-1 in patients with advanced solid tumors., Patients and Methods: We recruited patients with advanced solid tumors who failed standard therapy and received up to 3 prior lines of palliative systemic chemotherapy. TLD-1 was administered intravenously every 3 weeks up to a maximum of 9 cycles (6 for patients with prior anthracyclines) from a starting dose of 10 mg/m 2 , according to an accelerated titration design followed by a modified continual reassessment method., Results: 30 patients were enrolled between November 2018 and May 2021. No dose-limiting toxicities (DLT) were observed. Maximum administered dose of TLD-1 was 45 mg/m 2 , RP2D was defined at 40 mg/m 2 . Most frequent treatment-related adverse events (TRAE) of any grade included palmar-plantar erythrodysesthesia (PPE) (50% of patients), oral mucositis (50%), fatigue (30%) and skin rash (26.7%). Most common G3 TRAE included PPE in 4 patients (13.3%) and oral mucositis in 2 (6.7%). Overall objective response rate was 10% in the whole population and 23.1% among 13 patients with breast cancer; median time-to-treatment failure was 2.7 months. TLD-1 exhibit linear pharmacokinetics, with a median terminal half-life of 95 h., Conclusions: The new liposomal doxorubicin formulation TLD-1 showed a favourable safety profile and antitumor activity, particularly in breast cancer. RP2D was defined at 40 mg/m 2 administered every 3 weeks. (NCT03387917)., Competing Interests: Declaration of Competing Interest Ilaria Colombo: Travel grants: Tesaro, Janssen, AZ, GSK. Honoraria for consultancy or expert opinion: AZ, GSK, Novartis, MSD, BionTech. Institutional grants for clinical trials (PI): MSD, Bayer, Vivesto, Incyte, AZ, Orion. Kira-Lee Koster: Travel grants for congress visits from Takeda Pharma AG und Janssen-Cilag AG (paid to institution). Lisa Holer: no conflict of interest. Simon Haefliger: no conflict of interest. Manuela Rabaglio: no conflict of interest. Sara Bastian: no conflict of interest. Michael Schwitter: no conflict of interest. Katrin Eckhardt: no conflict of interest. Stefanie Hayoz: no conflict of interest. Anna M. Mc Laughlin: current employee of Pharmetheus AB and a paid consultant to multiple pharmaceutical companies. Charlotte Kloft: no conflict of interest. Marian Klose: no conflict of interest. Stefan Halbherr: Stefan Halbherr owns shares of InnoMedica Holding AG and functions as part of the executive management of the firm. Christian Baumgartner: Christian Baumgartner hold shares of Innomedica and is a former employee of the firm. He has received advisory fees from Boehringer Ingelheim. Cristiana Sessa: no conflict of interest. Anastasios Stathis: Institutional funding for clinical trials: Innomedica, Abbvie; ADC Therapeutics; Amgen, Astra Zeneca; Bayer; Cellestia; Incyte, Loxo Oncology; Merck MSD; Novartis; Pfizer; Philogen; Roche. Consultant/expert testimony/advisory board (institutional): Debiopharm, Janssen, AstraZeneca, Incyte, Eli Lilly, Novartis, Roche, Loxo Oncology. Travel grant: Incyte; Astra Zeneca. Dagmar Hess: no conflict of interest. Markus Joerger: Advisory role (institutional): Novartis, Astra Zeneca, Basilea Pharmaceutica, Bayer, BMS, Debiopharm, MSD, Roche, Sanofi; research funding: Swiss Cancer Research; travel grants: Roche, Sanofi, Takeda., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Despite recent awareness of institutional racism, there are still important racial disparities in prostate cancer medical research. We investigated the historical development of research on racial disparities and bias., Methods: PubMed was searched for the term 'prostate cancer race' and added key terms associated with racial disparity. As an indicator of scientific interest in the topic, we analyzed whether the number of publications increased linearly as an indicator of growing interest. The linearity is expressed as R 2 ., Results: The general search term "prostate cancer race" yielded 4507 publications. More specific search terms with ≥12 publications showing a higher scientific interest were found after 2005. The terms with the most publications when added to the general term were "genetic" (n = 1011), "PSA" (n = 995), and "detection" (n = 861). There was a linear increase in publications for "prostate cancer race" (R 2 = 0.75) since 1980. Specific terms added to the general terms with a high linear increase (R 2 ≥ 0.7) were "screening" (R 2 = 0.82), "detection" (R 2 = 0.72), "treatment access" (R 2 = 0.71), and "trial underrepresentation" (R 2 = 0.71). However, only a few studies have investigated its association with sexual activity. A combination with "sexual" showed 157 publications but only two years with ≥12 publications/year., Conclusion: The terms "genetic", "PSA", and "detection" have been the focus of recent research on racial differences in prostate cancer. We found that old stereotypes are still being mentioned but seem to find little interest in the current literature. Further research interest was found in "treatment access". Recently, interest in socioeconomic factors has decreased.

Perioperative anxiety is common. The relationship between anxiety and patient satisfaction with anaesthesia is still under debate. We assessed the prevalence and different causes of anaesthesia-related fears leading to perioperative anxiety and its association with patient satisfaction. A multiple-time validated, psychometrically developed questionnaire assessing the presence of anxiety, causes of fear, and different dimensions of patient satisfaction was sent to patients after discharge. The clinical data were obtained from a previous study. The sample size was calculated to recruit a minimum of 300 completed questionnaires. Statistical analyses included multivariate logistic regression models. Complete data were available for 474 of the 600 patients recruited for the study (response rate: 79%). A total of 141 patients (30%) reported anxiety regarding anaesthesia before hospital admission. The prevalence of anxiety was significantly associated with patient age (< 54 years: n = 196, prevalence = 37%; > 54 years: n = 263, prevalence = 24%; p = 0.002), female sex (female: n = 242, prevalence 39%; male: n = 223, prevalence 20%; p < 0.001), and surgical speciality (gynaecology (n = 61, prevalence = 49%), otolaryngology (n = 56, prevalence = 46%); p < 0.001). Fear of not awakening from anaesthesia (n = 44, prevalence = 32%, SD 45.8) and developing postoperative nausea or vomiting (n = 42, prevalence = 30%, SD 46.0) were the most reported anaesthesia-related causes of fear. Anxiety was associated with impaired overall patient satisfaction (mean dissatisfaction score 15%, versus 23%, SD 16.3 in the anxious group, SD 16.3, p < 0.001), especially regarding the dimensions "information and involvement in decision-making" (14% of deficits stated in the non-anxious group compared to 23% in the anxious group, p < 0.001), "respect and trust" (2% vs 6.26%, p < 0.001) and "continuity of care" (50% vs 57%, p < 0.015). Patient-reported anaesthesia-related anxiety is common and may affect important outcome parameters such as patient satisfaction. Abstract presented in e-poster format at Euroanaesthesia 2023, June 3-5, Glasgow., (© 2023. Springer Nature Limited.)

In myelofibrosis, comorbidities (CMs) add prognostic information independently from the Dynamic International Prognostic Scoring System (DIPSS). The Myelodysplastic Syndrome-Specific Comorbidity Index (MDS-CI) offers a simple tool for CM assessment as it is calculable after having performed a careful history and physical examination, a small routine chemistry panel (including creatinine and liver enzymes) and a limited set of functional diagnostics. To assess the prognostic impact of the MDS-CI in addition to the DIPSS and the Mutation-Enhanced International Prognostic Scoring System (MIPSS)-70, we performed a retrospective chart review of 70 MF patients who had not received allogeneic stem cell transplantation (primary MF, n = 51; secondary MF, n = 19; median follow-up, 40 months) diagnosed at our institution between 2000 and 2020. Cardiac diseases (23/70) and solid tumors (12/70) were the most common CMs observed at MF diagnosis. Overall survival (OS) was significantly influenced by the MDS-CI (median OS MDS-CI low (n = 38): 101 months; MDS-CI intermediate (n = 25): 50 months; and high (n = 7): 8 months; p < 0.001). The MDS-CI added prognostic information after inclusion as a categorical variable in a multivariate model together with the dichotomized DIPSS or the dichotomized MIPSS70: MDS-CI high HR 14.64 (95% CI 4.42; 48.48), p = 0.0002, and MDS-CI intermediate HR 1.97 (95% CI 0.96; 4.03), p = 0.065, and MDS-CI high HR 19.65 (95% CI 4.71; 81.95), p < 0.001, and MDS-CI intermediate HR 1.063 (95% CI 0.65; 4.06), p = 0.2961, respectively. The analysis of our small and retrospective MF cohort suggests that the MDS-CI represents a useful tool to identify MF patients with an increased vulnerability due to comorbidities. However, analyses of larger cohorts are necessary to define the value of the MDS-CI as a prognostic tool in comparison with other comorbidity indices.

Background: Up to now, no prospective cohort study using a validated questionnaire has assessed patients' expectation and perception of divided anesthesia care and its influence on patient satisfaction., Objective: We assessed patient satisfaction with divided anesthesia care in a district general hospital in Switzerland. We hypothesized that patient expectations, combined with their perceptions of the (un)importance of continuous anesthesia care would influence patient satisfaction., Material and Methods: A total of 484 eligible in-patients receiving anesthesia from October 2019 to February 2020 were included and received preoperative information about divided care via a brochure and face-to-face. The primary outcome was the assessment of patient satisfaction with divided anesthesia care using a validated questionnaire. In group 1 continuity of care was considered important but not performed. In group 2 continuity was ensured. In group 3 continuity was regarded as not important and was not performed. In group 4 patients could not remember or did not answer. A psychometrically developed validated questionnaire was sent to patients at home after discharge., Results: A total of 484 completed questionnaires (response rate 81%) were analyzed. In group 1 (n = 110) the mean total dissatisfaction score was 25% (95% confidence interval [CI] 21.8-28.1), in group 2 (n = 61) 6.8% (95% CI 4.8-8.7), in group 3 (n = 223) 12.1% (95% CI 10.7-13.4), and in group 4 (n = 90) 15% (95% CI 11-18); ANOVA: p < 0.001, η = 0.43. Of the patients 286 (59%) considered continuity of care by the same anesthetist relatively unimportant (34%) or not important at all (25%). The other 40% considered it important (22%) or very important (18%)., Conclusion: Despite receiving comprehensive preoperative information about divided anesthesia care, 40% of patients still considered continuity of care by the same anesthetist important. We recommend further research evaluating whether and how patient expectations can be modified towards the common practice of divided care and patient satisfaction can be increased., (© 2022. The Author(s).)

Antibody-drug conjugates (ADCs) have changed the treatment of breast cancer (BC) in more recent years. BC is a heterogenous group of malignancies with a broad range of histopathological characteristics. ADCs represent a class of therapeutics that combines an antigen-specific antibody backbone bound to a potent cytotoxic agent (the payload), via a linker, contributing to an improved therapeutic index. Currently, three ADCs received approval by the US Food and Drug Administration (FDA) and are in routine clinical use in different treatment settings; many more ADCs are in earlier and later stages of development, and their future approval will improve treatment options for patients with advanced but potentially also early-stage BC over time. Just recently, the results of three phase 3 trials (ASCENT, TULIP, and DESTINY-Breast03) evaluating sacituzumab govitecan (SG), trastuzumab duocarmazine, and trastuzumab deruxtecan (T-DXd) in different treatment settings were presented and showed promising results. This overview focuses on the newer ADCs, including T-DXd and SG, their pharmacology, mechanisms of action, and relevant studies. In addition, the latest results from trials investigating some newer ADCs, in further stages of development are presented., Competing Interests: KLK declares that she has no conflicts of interest. MJ is the investigator in Daiichi-Sankyo trials. JH receives research funding (top institution) from Celgene, Novartis, Hexal, Lilly; receives honoraria from Lilly, Novartis, Roche, Pfizer, AstraZeneca, MSD, Celgene, Eisai, Abbvie, Seagen, Gilead; has consulting advisory relationship with Lilly, Novartis, Roche, Pfizer, Hexal, AstraZeneca, MSD, Celgene, Abbvie, Seagen, Gilead; receives travel expenses from Roche, Pfizer, Novartis, Celgene, Daiichi., (© The Author(s) 2022.)

The moss Physcomitrella patens has been used as a model organism to study the induction of desiccation tolerance (DT), but links between dehydration rate, the accumulation of endogenous abscisic acid (ABA) and DT remain unclear. In this study, we show that prolonged acclimation of P. patens at 89% relative humidity (RH) [-16 MPa] can induce tolerance of desiccation at 33% RH (-153 MPa) in both protonema and gametophore stages. During acclimation, significant endogenous ABA accumulation occurred after 1 day in gametophores and after 2 days in protonemata. Physcomitrella patens expressing the ABA-inducible EARLY METHIONINE promoter fused to a cyan fluorescent protein (CFP) reporter gene revealed a mostly uniform distribution of the CFP increasing throughout the tissues during acclimation. DT was measured by day 6 of acclimation in gametophores, but not until 9 days of acclimation for protonemata. These results suggest that endogenous ABA accumulating when moss cells experience moderate water loss requires sufficient time to induce the changes that permit cells to survive more severe desiccation. These results provide insight for ongoing studies of how acclimation induces metabolic changes to enable DT in P. patens., (© 2018 Scandinavian Plant Physiology Society.)

The moss Physcomitrella patens, a model system for basal land plants, tolerates several abiotic stresses, including dehydration. We previously reported that Physcomitrella patens survives equilibrium dehydration to -13 MPa in a closed system at 91% RH. Tolerance of desiccation to water potentials below -100 MPa was only achieved by pretreatment with exogenous abscisic acid (ABA). We report here that gametophores, but not protonemata, can survive desiccation below -100 MPa after a gradual drying regime in an open system, without exogenous ABA. In contrast, faster equilibrium drying at 90% RH for 3-5 days did not induce desiccation tolerance in either tissue. Endogenous ABA accumulated in protonemata and gametophores under both drying regimes, so did not correlate directly with desiccation tolerance. Gametophores of a Ppabi3a/b/c triple knock out transgenic line also survived the gradual dehydration regime, despite impaired ABA signaling. Our results suggest that the initial drying rate, and not the amount of endogenous ABA, may be critical in the acquisition of desiccation tolerance. Results from this work will provide insight into ongoing studies to uncover the role of ABA in the dehydration response and the underlying mechanisms of desiccation tolerance in this bryophyte., (© 2017 John Wiley & Sons Ltd.)

Leishmaniasis is caused by different species of the protozoa, Leishmania, and frequently found in South-Western Asia, Eastern Africa, Brazil, and Mediterranean countries. Leishmania are transmitted to humans by the bite of sandflies. After weeks to months, unspecific symptoms may occur, accompanied by more specific findings like pancytopenia and organomegaly. We report two children with pancytopenia and hepato-/splenomegaly: a 1-year-old boy was first diagnosed with an Adenovirus-infection, accompanied by fever, pancytopenia, and hepatosplenomegaly who had spent his summer vacation in Spain and a 3-year-old boy of Macedonian origin who was first diagnosed with a Parvovirus B19-infection again accompanied by splenomegaly and pancytopenia. In both children, leukemia was excluded by an initial bone marrow puncture. As fever was still persistent weeks after the children's first hospital stay, both children received antibiotics empirically without sustainable effect. While different autoantibodies were present in both children, an immunosuppressive therapy was initiated in the younger boy without therapeutic success. A second bone marrow puncture was performed and Leishmania were finally detected morphologically and proven serologically. After weight-adjusted treatment with liposomal Amphotericin B for 10 days, both children recovered completely without relapse. Aim of this report is to broaden the spectrum of differential diagnoses in children with pancytopenia, splenomegaly, and fever to visceral leishmaniasis particularly when travel history is positive for the Mediterranean area. The infection may mimic more common diseases, such as leukemia, viral infections, or autoimmune diseases, because polyclonal B cell activation and other mechanisms may lead to multiple positive serologic tests. Both cases illustrate typical pitfalls and shall encourage taking Leishmaniasis into diagnostic consideration.

In response to the Howard Hughes Medical Institute/Association of American Medical Colleges Scientific Foundations for Future Physicians (SFFP) report and a concern for better preparing undergraduates for future doctoral programs in the health professions, the deans of the College of Arts and Sciences and Division of Basic Biomedical Sciences of Sanford School of Medicine of the University of South Dakota formed an ad hoc Premedical Curriculum Review Committee with representatives from the science departments and medical school. The Committee began by reviewing the university's suggested premedical curriculum and matching it to the proposed competencies from the SFFP to document duplications and deficiencies. The proposed changes in the Medical College Admission Test for 2015 were also evaluated. The Committee proposed a stronger premedical curriculum, with the development of some new courses, including an inquiry-based physiology course with team-based learning, to more fully address SFFP competencies. These analyses convinced the university that a new major would best help students achieve the competencies and prepare them for admission exams. Thus, a new Medical Biology major was proposed to the South Dakota Board of Regents and accepted for its initial offering in 2012. The new major has been broadly advertised to future students and is successful as a recruiting tool for the university. This article details the process of evaluating the curriculum and designing the new major, describes some of the difficulties in its implementation, and reviews outcomes from the new major to date., (Copyright © 2015 The American Physiological Society.)

Seed oils enriched in omega-7 monounsaturated fatty acids, including palmitoleic acid (16:1∆9) and cis-vaccenic acid (18:1∆11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega-7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ∆9 desaturation of stearoyl (18:0)-acyl carrier protein (ACP) to ∆9 desaturation of palmitoyl (16:0)-acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed-specific co-expression of a mutant ∆9-acyl-ACP and an acyl-CoA desaturase with high specificity for 16:0-ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega-7 monounsaturated fatty acids were obtained. Further increases in omega-7 fatty acid accumulation to 60-65% of the total fatty acids in camelina seeds were achieved by inclusion of seed-specific suppression of 3-keto-acyl-ACP synthase II and the FatB 16:0-ACP thioesterase genes to increase substrate pool sizes of 16:0-ACP for the ∆9-acyl-ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications., (© 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Medium-chain acyl-coenzyme-A dehydrogenase (MCAD) catalyzes the first step of mitochondrial beta-oxidation for medium-chain acyl-CoAs. Mutations in the ACADM gene cause MCAD deficiency presenting with life-threatening symptoms during catabolism. Since fatty-acid-oxidation disorders are part of newborn screening (NBS), many novel mutations with unknown clinical relevance have been identified in asymptomatic newborns. Eighteen of these mutations were separately cloned into the human ACADM gene, heterologously overexpressed in Escherichia coli and functionally characterized by using different substrates, molecular chaperones, and measured at different temperatures. In addition, they were mapped to the three-dimensional MCAD structure, and cross-link experiments were performed. This study identified variants that only moderately affect the MCAD protein in vitro, such as Y42H, E18K, and R6H, in contrast to the remaining 15 mutants. These three mutants display residual octanoyl-CoA oxidation activities in the range of 22 % to 47 %, are as temperature sensitive as the wild type, and reach 100 % activity with molecular chaperone co-overexpression. Projection into the three-dimensional protein structure gave some indication as to possible reasons for decreased enzyme activities. Additionally, six of the eight novel mutations, functionally characterized for the first time, showed severely reduced residual activities < 5 % despite high expression levels. These studies are of relevance because they classify novel mutants in vitro on the basis of their corresponding functional effects. This basic knowledge should be taken into consideration for individual management after NBS.

Scattering techniques have played a key role in our understanding of the structure and function of phospholipid membranes. These techniques have been applied widely to study how different molecules (e.g., cholesterol) can affect phospholipid membrane structure. However, there has been much less attention paid to the effects of molecules that remain in the aqueous phase. One important example is the role played by small solutes, particularly sugars, in protecting phospholipid membranes during drying or slow freezing. In this paper, we present new results and a general methodology, which illustrate how contrast variation small angle neutron scattering (SANS) and synchrotron-based X-ray scattering (small angle (SAXS) and wide angle (WAXS)) can be used to quantitatively understand the interactions between solutes and phospholipids. Specifically, we show the assignment of lipid phases with synchrotron SAXS and explain how SANS reveals the exclusion of sugars from the aqueous region in the particular example of hexagonal II phases formed by phospholipids.

Phase diagrams are presented for dipalmitoylphosphatidylcholine (DPPC) in the presence of sugars (sucrose) over a wide range of relative humidities (RHs). The phase information presented here, determined by small angle X-ray scattering (SAXS), is shown to be consistent with previous results achieved by differential scanning calorimetry (DSC). Both techniques show a significant effect of sucrose concentration on the phase behaviour of this phospholipid bilayer. An experimental investigation into the effect of sugars on the kinetic behaviour of the gel to fluid transition is also presented showing that increasing the sugar content appears to slightly increase the rate at which the transition occurs.

Dehydration tolerance in plants is an important but understudied component of the complex phenotype of drought tolerance. Most plants have little capacity to tolerate dehydration; most die at leaf water potentials between -5 and -10 MPa. Some of the non-vascular plants and a small percentage (0.2%) of vascular plants, however, can survive dehydration to -100 MPa and beyond, and it is from studying such plants that we are starting to understand the components of dehydration tolerance in plants. In this chapter we define what dehydration tolerance is and how it can be assessed, important prerequisites to understanding the response of a plant to water loss. The metabolic and mechanical consequences of cellular dehydration in plants prelude a discussion on the role that gene expression responses play in tolerance mechanisms. We finally discuss the key biochemical aspects of tolerance focusing on the roles of carbohydrates, late embryogenesis abundant and heat shock proteins, reactive oxygen scavenging (ROS) pathways, and novel transcription factors. It is clear that we are making significant advances in our understanding of dehydration tolerance and the added stimulus of new model systems will speed our abilities to impact the search for new strategies to improve drought tolerance in major crops.

We present an X-ray scattering study of the effects of dehydration on the bilayer and chain-chain repeat spacings of dipalmitoylphosphatidylcholine bilayers in the presence of sugars. The presence of sugars has no effect on the average spacing between the phospholipid chains in either the fluid or gel phase. Using this finding, we establish that for low sugar concentrations only a small amount of sugar exclusion occurs. Under these conditions, the effects of sugars on the membrane transition temperatures can be explained quantitatively by the reduction in hydration repulsion between bilayers due to the presence of the sugars. Specific bonding of sugars to lipid headgroups is not required to explain this effect.

We present a quantitative study of the effect of sugars on the membrane gel-fluid phase transition as a function of sugar:lipid ratio. We show that the maximum effect occurs at around 1.5 sugar rings per molecule for both mono- and di-saccharides. We present a theoretical model to try to explain these results, and discuss the assumptions inherent in the model.

Desiccation tolerance of protoplasts isolated from germinating pea (Pisum sativum L. cv. 'Alaska') embryonic axes depends, in part, on the osmotic strength and composition of the suspending medium. To determine the reason for this dependence and whether treatment with different solutions results in different types of damage, protoplast recovery and survival were assessed after dehydration to a range of water contents. Protoplasts were derived from germinating axes that had intermediate desiccation tolerance. Protoplasts were isolated and resuspended in buffers containing sucrose/raffinose (85:15, w/w) or sorbitol, which were isotonic or hypertonic to the cells of the embryonic axis, then were flash-dried to a range of water contents. Protoplasts were rehydrated and stained with fluorescein diacetate (FDA) to assess survival and to estimate two types of membrane injury: lysis and the loss of semipermeability. In all treatments, protoplast survival dropped sharply during the initial phase of dehydration due to lysis. Protoplast survival was greater in hypertonic sucrose/raffinose buffer than in isotonic sucrose/raffinose buffer, or in the latter made hypertonic by the addition of sorbitol. When sorbitol was substituted for sucrose/raffinose in either the isolation or desiccation buffer, or both, protoplast survival at intermediate and low hydrations decreased due to a loss of membrane semipermeability. The results indicate that additional sucrose/raffinose is beneficial for the desiccation tolerance of protoplasts, the benefit is not due to a simple osmotic effect, and the benefit is greatest at water contents less than 0.5 g g(-1) DW, where the presence of the sugars appears to protect membrane semipermeability.

The teaching faculty for this course sought to address their own concerns about the quality of student learning in an impersonal large lecture biology class for majors, the difficulties in getting to know each student by name, and difficulties in soliciting answers and reactions from the students during the lecture. Questions addressed by this study were, Do active-learning activities in a small and personal lecture setting enhance student learning more than active-learning activities in large impersonal lectures? and Are students more satisfied with an educational experience in a small and personal lecture setting? Based on faculty perceptions of how they best relate to their students, the prediction was that the students in the experimental group with small lecture classes and increased direct contact with the teaching faculty would learn physiological principles better than the students in the control group in the large impersonal lecture portion of the course. One of the laboratory sections of this large enrollment biology course was randomly selected to be taught with separate small lectures by the teaching faculty. In addition, the teaching faculty participated in the laboratory with these students during their experiments correlated with the lecture material. The students in both groups were compared by pre- and posttests of physiological principles, final course grades, and class satisfaction surveys.

Sorghum plants inhibit the growth of some adjacent species. Root exudates from grain sorghum (Sorghum bicolor), consisting primarily of the quinone sorgoleone, are phytotoxic to several plant species, yet the mechanisms of growth inhibition remain to be fully explained. Disruption of electron transport functions in isolated mitochondria and chloroplasts has been reported as one explanation for growth inhibition. In the studies reported here, however, soybean seedlings grown in nutrient solution with 10, 50, or 100 microM sorgoleone showed no disruption of photosynthesis, as measured by leaf fluorescence and oxygen evolution, yet their mean leaf surface area was less when grown in 100 microM sorgoleone. Furthermore, in the presence of these same concentrations of sorgoleone, decreased nutrient solution use by soybean seedlings and decreased H+-ATPase activity in corn root microsomal membranes were observed. This suggests that impairment of essential plant processes, such as solute and water uptake, driven by proton-pumping across the root cell plasmalemma should also be considered as a mechanism contributing to observed plant growth inhibition by sorgoleone.

Sorption isotherms were obtained for a range of lipid/sugar/water mixtures. These were analysed using a simple hydration forces formalism. The results demonstrate that this simple analysis can be used to estimate dehydration parameters for these relatively complex systems. This in turn provides some insight into the location and role of sugars in the hydration behaviour of lipid systems. The relevance of these results to the phase behaviour of lipid/sugar mixtures during dehydration are discussed.

Juglone is phytotoxic, but the mechanisms of growth inhibition have not been fully explained. Previous studies have proposed that disruption of electron transport functions in mitochondria and chloroplasts contribute to observed growth reduction in species exposed to juglone. In studies reported here, corn and soybean seedlings grown in nutrient solution amended with 10, 50, or 100 microM juglone showed significant decreases in root and shoot dry weights and lengths with increasing concentrations. However, no significant differences in leaf chlorophyll fluorescence or CO2-dependent leaf oxygen evolution were observed, even in seedlings that were visibly affected. Disruption of root oxygen uptake was positively correlated with increasing concentrations of juglone, suggesting that juglone may reach mitochondria in root cells. Water uptake and acid efflux also decreased for corn and soybean seedlings treated with juglone, suggesting that juglone may affect metabolism of root cells by disrupting root plasma membrane function. Therefore, the effect of juglone on H+-ATPase activity in corn and soybean root microsomes was tested. Juglone treatments from 10 to 1000 microM significantly reduced H+-ATPase activity compared to controls. This inhibition of H+-ATPase activity and observed reduction of water uptake offers a logical explanation for previously documented phytotoxicity of juglone. Impairment of this enzyme's activity could affect plant growth in a number of ways because proton-pumping in root cells drives essential plant processes such as solute uptake and, hence, water uptake.

Protoplasts were isolated from pea (Pisum sativum L. cv. Alaska) embryonic axes during and after germination to determine whether the loss of desiccation tolerance in the embryos also occurs in the protoplasts. At all times studied, protoplast survival decreased as water content decreased; however, the sensitivity to dehydration was less when the protoplasts were isolated from embryos that were still desiccation-tolerant (12 h and 18 h of imbibition) than when protoplasts were derived from axes that were sensitive (24 h and 36 h of imbibition). The water content at which 50% of the population was killed (WC50) increased throughout germination and early seedling growth for both the intact tissue and the protoplasts derived from them. Prior to radicle emergence, protoplasts were less desiccation-tolerant than the intact axes; however, protoplasts isolated from radicles shortly after emergence had lower WC50s than the intact radicles. A comparison of protoplast survival after isolation and dehydration in either 500 mM sucrose/raffinose or 700 mM sucrose revealed no difference in tolerance except at 24 h of imbibition, when protoplasts treated in the more concentrated solution had improved tolerance of dehydration. Although intact epicotyls are generally more desiccation-tolerant than radicles, protoplasts isolated separately from epicotyls and radicles did not differ in tolerance. Collectively, these data suggest that protoplasts gradually lose desiccation tolerance during germination, as do the orthodox embryos from which they were derived. However, even prior to radicle emergence, protoplasts display a sensitivity to progressive dehydration that is similar to that shown by recalcitrant and ageing embryos.

The effect of increasing solute size on phosphatidylcholine phase behaviour at a range of hydrations was investigated using differential scanning calorimetry. Dehydration of phospholipid membranes gives rise to a compressive stress within the bilayers that promotes fluid-to-gel phase transitions. According to the Hydration Forces Explanation, sugars in the intermembrane space minimize the compressive stress and limit increases in the fluid-gel transition temperature, T(m), by acting as osmotic and volumetric spacers that hinder the close approach of membranes. However, the sugars must remain between the bilayers in order to limit the rise in T(m). Large polymers are excluded from the interlamellar space during dehydration and do not limit the dehydration-induced rise in T(m). In this study, we used maltodextrins with a range of molecular weights to investigate the size-exclusion limit for polymers between phosphatidylcholine bilayers. Solutes with sizes ranging from glucose to dextran 1000 limited the rise in lipid T(m) during dehydration, suggesting that they remain between dehydrated bilayers. At the lowest hydrations the solutions vitrified, and T(m) was further depressed to about 20 degrees C below the transition temperature for the lipid in excess water, T(o). The depression of T(m) below T(o) occurs when the interlamellar solution vitrifies between fluid phase bilayers. The larger maltodextrins, dextran 5000 and 12,000, had little effect on the T(m) of the PCs at any hydration, nor did vitrification of these larger polymers affect the lipid phase behaviour. This suggests that the larger maltodextrins are excluded from the interlamellar region during dehydration.

Water that remains unfrozen at temperatures below the equilibrium bulk freezing temperature, in the presence of ice, is sometimes called unfreezable or bound. This paper analyses the phenomenon in terms of quantitative measurements of the hydration interaction among membranes or macromolecules at freezing temperatures. These results are related to analogous measurements in which osmotic stress or mechanical compression is used to equilibrate water of hydration with a bulk phase. The analysis provides formulas to estimate, at a given sub-freezing temperature, the amount of unfrozen water due to equilibrium hydration effects. Even at tens of degrees below freezing, this hydration effect alone can explain an unfrozen water volume that considerably exceeds that of a single 'hydration shell' surrounding the hydrophilic surfaces. The formulas provided give a lower bound to the amount of unfrozen water for two reasons. First, the well-known freezing point depression due to small solutes is, to zeroth order, independent of the membrane or macromolecular hydration effect. Further, the unfrozen solution found between membranes or macromolecules at freezing temperatures has high viscosity and small dimensions. This means that dehydration of such systems, especially at freezing temperatures, takes so long that equilibrium is rarely achieved over normal experimental time scales. So, in many cases, the amount of unfrozen water exceeds that expected at equilibrium, which in turn usually exceeds that calculated for a single hydration shell.

To facilitate studies of desiccation tolerance at the cellular level, a technique to isolate protoplasts from desiccation-tolerant pea (Pisum sativum L. cv. Alaska) embryos has been developed. Using FDA (fluorescein diacetate) as a probe, viability of the protoplasts was investigated before and after drying to determine whether the protoplasts could survive desiccation in a manner similar to the tissue from which they were isolated. Protoplasts were isolated from 12 h imbibed pea axes, suspended in several different sugar solutions, then dried to water contents less than 0.2 g H(2)O g(-1) DW. Protoplasts only survived drying if the rate was rapid (<2 h), while slow drying (24 h) was lethal. Maximal survival (75%) was obtained after drying protoplasts with a mixture of sucrose and raffinose, while pure sucrose and trehalose were somewhat less effective protectants. Low survival was obtained after drying protoplasts with monosaccharides and pure raffinose. Protoplasts isolated from germinated seedlings did not survive dehydration below 0.2 g H(2)O g(-1) DW. Transmission electron microscopy revealed that dried desiccation-tolerant protoplasts appeared shrunken, with folded membranes, while dried protoplasts from sensitive tissue had disrupted membranes. While isolated protoplasts maintained some of the desiccation tolerance of orthodox seeds, their inability to survive complete drying and their sensitivity to drying rate is similar to the behaviour of recalcitrant embryos.

DSC was used to study the ability of glass-forming sugars to affect the gel-to-fluid phase transition temperature, T(m), of several phosphatidylcholines during dehydration. In the absence of sugars, T(m) increased as the lipid dried. Sugars diminished this increase, an effect we explain using the osmotic and volumetric properties of sugars. Sugars vitrifying around fluid phase lipids lowered T(m) below the transition temperature of the fully hydrated lipid, T(o). The extent to which T(m) was lowered below T(o) ranged from 12 degrees to 57 degrees, depending on the lipids' acyl chain composition. Sugars vitrifying around gel phase lipids raised T(m) during the first heating scan in the calorimeter, then lowered it below T(o) in subsequent scans of the sample. Ultrasound measurements of the mechanical properties of a typical sugar-glass indicate that it is sufficiently rigid to hinder the lipid gel-to-fluid transition. The effects of vitrification on T(m) are explained using the two-dimensional Clausius-Clapeyron equation to model the mechanical stress in the lipid bilayer imposed by the glassy matrix. Dextran and polyvinylpyrrolidone (PVP) also vitrified but did not depress T(m) during drying. Hydration data suggest that the large molecular volumes of these polymers caused their exclusion from the interbilayer space during drying.

Increases in liver glycogen phosphorylase activity, along with inhibition of glycogen synthetase and phosphofructokinase-1, are associated with elevated cryoprotectant (glucose) levels during freezing in some freeze-tolerant anurans. In contrast, freeze-tolerant chorus frogs, Pseudacris triseriata, accumulate glucose during freezing but exhibit no increase in phosphorylase activity following 24-h freezing bouts. In the present study, chorus frogs were frozen for 5- and 30-min and 2- and 24-h durations. After freezing, glucose, glycogen, and glycogen phosphorylase and synthetase activities were measured in leg muscle and liver to determine if enzyme activities varied over shorter freezing durations, along with glucose accumulation. Liver and muscle glucose levels rose significantly (5-12-fold) during freezing. Glycogen showed no significant temporal variation in liver, but in muscle, glycogen was significantly elevated after 24 h of freezing relative to 5 and 30 min-frozen treatments. Hepatic phosphorylase a and total phosphorylase activities, as well as the percent of the enzyme in the active form, showed no significant temporal variation following freezing. Muscle phosphorylase a activity and percent active form increased significantly after 24 h of freezing, suggesting some enhancement of enzyme function following freezing in muscle. However, the significance of this enhanced activity is uncertain because of the concurrent increase in muscle glycogen with freezing. Neither glucose 6-phosphate independent (I) nor total glycogen synthetase activities were reduced in liver or muscle during freezing. Thus, chorus frogs displayed typical cryoprotectant accumulation compared with other freeze-tolerant anurans, but freezing did not significantly alter activities of hepatic enzymes associated with glycogen metabolism.

Mechanistic bases for freezing tolerance in anurans have been well-studied only in wood frogs, Rana sylvatica, so comprehensive explanations for the mechanisms and evolution of freezing tolerance in anurans are lacking. We measured crystallization temperatures, freezing tolerance/intolerance, and tissue glucose and glycogen phosphorylase activities in frozen and unfrozen winter-acclimated Pseudacris triseriata, Bufo cognatus and B. woodhousei. Freezing occurred at higher subzero temperatures on wet substrate than on dry substrate in all species, indicating susceptibility to inoculative freezing. P. triseriata was freeze-tolerant, but survival was dependent on the level of supercooling prior to freezing. All Bufo were freezing intolerant, regardless of crystallization temperature. Glucose was significantly elevated by freezing in both liver (35-fold) and leg muscle (22-fold) in winter P. triseriata, but only liver glucose was significantly elevated in B. cognatus. However, freezing did not alter glycogen phosphorylase activity in either species. Liver phosphorylase activity was significantly higher in P. triseriata than in B. cognatus, suggesting that capacity for mobilizing glucose from liver glycogen is associated with freezing tolerance. Summer measurements of liver phosphorylase activity, however, did not differ between species. Thus, P. triseriata, but not B. cognatus, exhibited winter increment of liver phosphorylase activity that is correlated with the development of freezing tolerance.

The phase behavior of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) was characterized as a function of hydration in the presence of combinations of sugars representative of sugars found in seed embryos having differing degrees of desiccation tolerance. The tendency of the sugar mixes to vitrify was also monitored as a function of hydration. Using differential scanning calorimetry, it was found that all sugars diminished the increase in the gel-to-fluid phase transition temperature (Tm) of POPC that occurred upon dehydration of the pure lipid. These results are analyzed in terms of the osmotic and volumetric properties of sugars. Also, it was found that in those samples for which the glass transition temperature (Tg) was greater than the Tm of POPC, Tm was lowered by approx. 20 C degrees from the value for the fully hydrated lipid. X-ray diffraction data confirmed that acyl chain freezing was deferred to a lower temperature during cooling of vitrified samples. The significance of these results is discussed in terms of the ability of many organisms to tolerate desiccation.

During cold acclimation of Puma rye (Secale cereale L. cv Puma), the intracellular osmotic potential nearly doubles. During this period, the accumulation of glycinebetaine, proline, and soluble sugars was monitored. The amount of glycinebetaine increased from 290 to 1300 micrograms per gram fresh weight during the 4-week acclimation period. Proline content did not change during the first 3 weeks of acclimation but then increased from 27 to 580 micrograms per gram fresh weight during the next 3 weeks. The total soluble sugar content more than doubled by the second week of cold acclimation, increasing from 11 to 26 milligrams per gram fresh weight. Most of this increase can be attributed to the accumulation of sucrose and raffinose, whose levels increased from 2.4 and 0 to 11 and 5 milligrams per gram fresh weight, respectively. The content of monosaccharides, predominantly glucose, remained at a constant 10 milligrams per gram fresh weight throughout the acclimation period. A comparison of the sugar content of protoplasts versus vacuoles isolated from cold-acclimated leaves revealed that the extravacuolar volume contained monosaccharides, sucrose, and raffinose. Thus, the increased amounts of sucrose and raffinose that occur during cold acclimation are present in compartments external to the vacuole and may contribute to cryoprotection.

The formation of intracellular glass may help protect embryos from damage due to desiccation. Soluble sugars similar to those found in desiccation tolerant embryos were studied with differential scanning calorimetry. Those sugars from desiccation tolerant embryos can form glasses at ambient temperatures, whereas those from embryos that do not tolerate desiccation only form glasses at subzero temperatures. It is concluded that tolerant embryo cells probably contain sugar glasses at storage temperatures and water contents, but intolerant embryo cells probably do not.

Soluble sugars have been shown to protect liposomes and lobster microsomes from desiccation damage, and a protective role has been proposed for them in several anhydrous systems. We have studied the relationship between soluble sugar content and the loss of desiccation tolerance in the axes of germinating soybean (Glycine max L. Merr. cv Williams), pea (Pisum sativum L. cv Alaska), and corn (Zea mays L. cv Merit) axes. The loss of desiccation tolerance during imbibition was monitored by following the ability of seeds to germinate after desiccation following various periods of preimbibition and by following the rates of electrolyte leakage from dried, then rehydrated axes. Finally, we analyzed the soluble sugar contents of the axes throughout the transition from desiccation tolerance to intolerance. These analyses show that sucrose and larger oligosaccharides were consistently present during the tolerant stage, and that desiccation tolerance disappeared as the oligosaccharides were lost. The results support the idea that sucrose may serve as the principal agent of desiccation tolerance in these seeds, with the larger oligosaccharides serving to keep the sucrose from crystallizing.