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Background: There is an escalating concern for the mental health of university students being recognized as a high-risk group for psychological distress. Despite research emphasizing the need to integrate mental well-being into higher education, existing interventions primarily focus on challenges and support services, leaving a gap in practical insights for promoting mental well-being at the university as a whole., Objectives: This paper aims to cover the theoretical and methodological foundations for the design and development of a complex multi-level intervention called the ABCs of mental health at the university (ABC-uni)., Methods: Following the MRC framework for complex interventions, the design and development of a novel complex intervention is described (Phase I). Using the socio-ecological model and incorporating principles from health promotion charters, capacity building, organizational change models, and the principles of the ABCs of mental health a program theory for mental health promotion at the university is proposed. Following this theoretical foundation a logic model outlines the ABC-uni intervention components at structural, community, and individual levels. The components include staff training, campaign materials, a photovoice project, integration of mental well-being literacy into introductory programs, a credit-bearing course, and an online module. Preferably, most of these components are developed and carried out collaboratively with students at the university. As proposed in the logic model expected outcomes encompass heightened awareness, supportive environments, commitment, and accountability, aiming to enhance mental well-being across the entire university. The design and development of the intervention components occur at a Danish public university., Discussion: This section addresses the strengths and limitations of the design of the ABC-uni intervention. Future research will cover the feasibility phase of each components of the ABC-uni intervention (phase II). The conceptual framework and program theory outlining mental health promotion at the university, along with the detailed description of the intervention components, provides valuable insights for fostering mental well-being in the university community., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Nielsen, Bermejo-Martins, Nelausen, Madsen, Riva, Koushede and Meilstrup.)

Superparamagnetic iron oxide nanoparticles (SPIONs) are used as contrast agents in magnetic resonance imaging (MRI) and magnetic particle imaging (MPI), and resulting images can be used to guide magnetothermal heating. Alternating magnetic fields (AMF) cause local temperature increases in regions with SPIONs, and we investigated the ability of magnetic hyperthermia to regulate temperature-sensitive repressors (TSRs) of bacterial transcription. The TSR, TlpA39, was derived from a Gram-negative bacterium and used here for thermal control of reporter gene expression in Gram-positive, Bacillus subtilis. In vitro heating of B. subtilis with TlpA39 controlling bacterial luciferase expression resulted in a 14.6-fold (12 hours; h) and 1.8-fold (1 h) increase in reporter transcripts with a 10.0-fold (12 h) and 12.1-fold (1 h) increase in bioluminescence. To develop magnetothermal control, B. subtilis cells were coated with three SPION variations. Electron microscopy coupled with energy dispersive X-ray spectroscopy revealed an external association with, and retention of, SPIONs on B. subtilis . Furthermore, using long duration AMF we demonstrated magnetothermal induction of the TSRs in SPION-coated B. subtilis with a maximum of 5.6-fold increases in bioluminescence. After intramuscular injections of SPION-coated B. subtilis , histology revealed that SPIONs remained in the same locations as the bacteria. For in vivo studies, 1 h of AMF is the maximum exposure due to anesthesia constraints. Both in vitro and in vivo , there was no change in bioluminescence after 1 h of AMF treatment. Pairing TSRs with magnetothermal energy using SPIONs for localized heating with AMF can lead to transcriptional control that expands options for targeted bacteriotherapies.

Developing modular tools that direct mammalian cell function and activity through controlled delivery of essential regulators would improve methods of guiding tissue regeneration, enhancing cellular-based therapeutics and modulating immune responses. To address this challenge, Bacillus subtilis was developed as a chassis organism for engineered endosymbionts (EES) that escape phagosome destruction, reside in the cytoplasm of mammalian cells, and secrete proteins that are transported to the nucleus to impact host cell response and function. Two synthetic operons encoding either the mammalian transcription factors Stat-1 and Klf6 or Klf4 and Gata-3 were recombined into the genome of B. subtilis expressing listeriolysin O (LLO) from Listeria monocytogenes and expressed from regulated promoters. Controlled expression of the mammalian proteins from B. subtilis LLO in the cytoplasm of J774A.1 macrophage/monocyte cells altered surface marker, cytokine and chemokine expression. Modulation of host cell fates displayed some expected patterns towards anti- or pro-inflammatory phenotypes by each of the distinct transcription factor pairs with further demonstration of complex regulation caused by a combination of the EES interaction and transcription factors. Expressing mammalian transcription factors from engineered intracellular B. subtilis as engineered endosymbionts comprises a new tool for directing host cell gene expression for therapeutic and research purposes., (© 2022. The Author(s).)

Superparamagnetic iron oxide nanoparticles (SPIONs) can be used as imaging agents to differentiate between normal and diseased tissue or track cell movement. Magnetic particle imaging (MPI) detects the magnetic properties of SPIONs, providing quantitative and sensitive image data. MPI performance depends on the size, structure, and composition of nanoparticles. Magnetotactic bacteria produce magnetosomes with properties similar to those of synthetic nanoparticles, and these can be modified by mutating biosynthetic genes. The use of Magnetospirillum gryphiswaldense , MSR-1 with a mamJ deletion, containing clustered magnetosomes instead of typical linear chains, resulted in improved MPI signal and resolution. Bioluminescent MSR-1 with the mamJ deletion were administered into tumor-bearing and healthy mice. In vivo bioluminescence imaging revealed the viability of MSR-1, and MPI detected signals in livers and tumors. The development of living contrast agents offers opportunities for imaging and therapy with multimodality imaging guiding development of these agents by tracking the location, viability, and resulting biological effects.

Objectives/hypothesis: Develop a patient-specific tissue engineered construct for laryngeal reconstruction following a partial laryngectomy., Study Design: Bench and animal research., Methods: A construct made from a porous polyethylene scaffold shaped in a canine-specific configuration and seeded with autologous canine adipose-derived stem cells in fibrin glue was implanted in a canine following a partial laryngectomy. After 1 year, the construct was first evaluated in vivo with high-speed imaging and acoustic-aerodynamic measures. It was then explanted and evaluated histologically., Results: The canine study at 1 year revealed the construct provided voicing (barking) with acoustic and aerodynamic measures within normal ranges. The canine was able to eat and breathe normally without long-term support. The construct was integrated with epithelialization of all areas except the medial portion of the vocal fold structure. No anti-infective agents were needed after the standard perioperative medications were completed., Conclusion: This study provided a successful first step toward developing a patient-specific composite construct for patients undergoing partial laryngectomies., Level of Evidence: NA Laryngoscope, 132:S1-S11, 2022., (© 2022 The American Laryngological, Rhinological and Otological Society, Inc.)

A glucose responsive insulin (GRI) that responds to changes in blood glucose concentrations has remained an elusive goal. Here we describe the development of glucose cleavable linkers based on hydrazone and thiazolidine structures. We developed linkers with low levels of spontaneous hydrolysis but increased level of hydrolysis with rising concentrations of glucose, which demonstrated their glucose responsiveness in vitro. Lipidated hydrazones and thiazolidines were conjugated to the LysB29 side-chain of HI by pH-controlled acylations providing GRIs with glucose responsiveness confirmed in vitro for thiazolidines. Clamp studies showed increased glucose infusion at hyperglycemic conditions for one GRI indicative of a true glucose response. The glucose responsive cleavable linker in these GRIs allow changes in glucose levels to drive the release of active insulin from a circulating depot. We have demonstrated an unprecedented, chemically responsive linker concept for biopharmaceuticals., (© 2020 Wiley-VCH GmbH.)

Competing Interests: Declaration of Competing Interest None.

Shewanella oneidensis MR-1 is quickly becoming a synthetic biology workhorse for bioelectrochemical technologies due to a high level of understanding of its interaction with electrodes. Transmembrane electron transfer via the Mtr pathway has been well characterized, however, the role of NADH dehydrogenases in feeding electrons to Mtr has been only minimally studied in S. oneidensis MR-1. Four NADH dehydrogenases are encoded in the genome, suggesting significant metabolic flexibility in oxidizing NADH under a variety of conditions. A strain lacking the two dehydrogenases essential for aerobic growth exhibited a severe growth defect with an anode (+0.4 V SHE ) or Fe(III)-NTA as the terminal electron acceptor. Our study reveals that the same NADH dehydrogenase complexes are utilized under oxic conditions or with a high potential anode. Our study also supports the previously indicated importance of pyruvate dehydrogenase activity in producing NADH during anerobic lactate metabolism. Understanding the role of NADH in extracellular electron transfer may help improve biosensors and give insight into other applications for bioelectrochemical systems.

Objective: To determine symptoms and characteristics of chronic sensory neuropathy in patients treated with oxaliplatin and docetaxel, including patterns of somatosensory abnormalities, pain descriptors, and psychological functioning., Design: A retrospective cross-sectional study., Setting: A chronic pain research center., Subjects: Thirty-eight patients with chronic peripheral pain and/or dysesthesia following chemotherapy., Methods: Sensory profiles, psychological functioning, and quality of life were assessed using standardized questionnaires. In addition, standardized quantitative sensory testing and nerve conduction studies were carried out., Results: The sensory profiles and clinical symptoms were very similar in the two groups. Pricking, numbness, and burning were common descriptors in both groups, and the predominant finding was sensory loss to A beta-mediated sensory modalities with decreased mechanical and vibration detection thresholds. A high frequency of abnormalities in thermal sensory limen and the presence of paradoxical heat sensation seem to be sensitive markers of small fiber loss. Both groups had mainly sensory, axonal large fiber or mixed fiber polyneuropathy, which tended to be most severe in the oxaliplatin group., Conclusions: Both oxaliplatin-induced and docetaxel-induced polyneuropathies represent a significant problem that affects the daily life of the patients. Our results, defining the somatosensory phenotype, can improve the understanding of the pathophysiological mechanisms useful for future studies in the tailored treatment of prevention of chemotherapy-induced peripheral neuropathy and pain.

A fully synthetic MUC1-based cancer vaccine was designed and chemically synthesized containing an endogenous helper T-epitope (MHC class II epitope). The vaccine elicited robust IgG titers that could neutralize cancer cells by antibody-dependent cell-mediated cytotoxicity (ADCC). It also activated cytotoxic T-lymphocytes. Collectively, the immunological data demonstrate engagement of helper T-cells in immune activation. A synthetic methodology was developed for a penta-glycosylated MUC1 glycopeptide, and antisera of mice immunized by the new vaccine recognized such a structure. Previously reported fully synthetic MUC1-based cancer vaccines that elicited potent immune responses employed exogenous helper T-epitopes derived from microbes. It is the expectation that the use of the newly identified endogenous helper T-epitope will be more attractive, because it will activate cognate CD4 + T-cells that will provide critical tumor-specific help intratumorally during the effector stage of tumor rejection and will aid in the generation of sustained immunological memory., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Polyglutamine (PolyQ) diseases are progressive neurodegenerative disorders caused by both protein- and RNA-mediated toxicities. We previously showed that a peptidyl inhibitor, P3, which binds directly to expanded CAG RNA can inhibit RNA-induced nucleolar stress and suppress RNA-induced neurotoxicity. Here we report a N-acetylated and C-amidated derivative of P3, P3V8, that showed a more than 20-fold increase in its affinity for expanded CAG RNA. The P3V8 peptide also more potently alleviated expanded RNA-induced cytotoxicity in vitro, and suppressed polyQ neurodegeneration in Drosophila with no observed toxic effects. Further N-palmitoylation of P3V8 (L1P3V8) not only significantly improved its cellular uptake and stability, but also facilitated its systemic exposure and brain uptake in rats via intranasal administration. Our findings demonstrate that concomitant N-acetylation, C-amidation and palmitoylation of P3 significantly improve both its bioactivity and pharmacological profile. L1P3V8 possesses drug/lead-like properties that can be further developed into a lead inhibitor for the treatment of polyQ diseases.

DNA-templated silver nanoclusters of a few tens of atoms or less have come into prominence over the last several years due to very strong absorption and efficient emission. Applications in microscopy and sensing have already been realized, however little is known about the excited-state structure and dynamics in these clusters. Here we report on a multidimensional spectroscopy investigation of the energy-level structure and the early-time relaxation cascade, which eventually results in the population of an emitting state. We find that the ultrafast intramolecular relaxation is strongly coupled to a specific vibrational mode, resulting in the concerted transfer of population and coherence between excited states on a sub-100 fs timescale.

Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides that target the BRAF V600E oncogene, with a library of rationally designed peptides employing CuAAC "click" chemistry. The peptide sequence has an influence on the specificity and affinity of target DNA/RNA binding. We also investigated the impact of locked nucleic acids (LNAs) on the latter. Lysine residues improve binding of POCs to target DNA and RNA, whereas the distance to lysine correlates exclusively with a decrease in binding of mismatched RNA targets. Glycine and tyrosine residues affect target binding as well. Importantly, the resistance of POCs to enzymatic degradation is dramatically improved by the internal attachment of peptides but not by LNA alone. Independently of the peptide sequence, the conjugates are stable for up to 24 h in 90% human serum and duplexes of POCs with complementary DNA for up to 160 h in 90% human serum. Such excellent stability has not been previously reported for DNA and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media.

Background: Persistent pain is frequent after thoracotomy, with a reported prevalence of up to 60%. It remains unclear why some patients develop pain, whereas others do not. We therefore examined patients with and without pain after thoracotomy to identify pathophysiological contributors to persistent pain., Methods: Twenty patients with persistent pain, 12 patients without pain and 20 healthy controls underwent detailed functional and structural assessment including psychometric and neuropathic pain questionnaires, bedside examination for pinprick hyperalgesia and brush allodynia, quantitative sensory testing according to the protocol of the German Research Network on Neuropathic Pain, measurement of capsaicin-evoked flare response, intradermal nerve density as determined by skin biopsies and laser- and heat-evoked potentials., Results: Bedside testing revealed evoked pain in 16 of 20 patients with pain, but only in 2 of 12 patients without pain (p < 0.001). Quantitative sensory testing showed increased mechanical pain sensitivity (p = 0.018) on the operated side in patients with pain, but there were no differences between the two patient groups with regard to intradermal nerve fibre density, area and flux following capsaicin application and laser- and heat-evoked potentials., Conclusion: Different and individual pathophysiological mechanisms of pain may obscure the clinical picture and thus preclude identification of a specific pain profile in patients with persistent post-thoracotomy pain., Significance: Evoked pain is more frequent in patients with pain. Assessment of intradermal nerve density, capsaicin-induced flare response and contact and laser heat-evoked potentials revealed no differences between pain patients and pain-free patients., (© 2016 European Pain Federation - EFIC®.)

Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide-oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design.

Objective: Clinical studies have found that patients with Alzheimer's disease report pain of less intensity and with a lower affective response, which has been thought to be due to altered pain processing. The authors wished to examine the cerebral processing of non-painful and painful stimuli using somatosensory evoked potentials and contact heat evoked potentials in patients with Alzheimer's disease and in healthy elderly controls., Design: Case-control study, Setting and Subjects: Twenty outpatients with mild-moderate Alzheimer's disease and in 17 age- and gender-matched healthy controls were included, Method: Contact heat evoked potentials and somatosensory evoked potentials were recorded in all subjects. Furthermore, warmth detection threshold and heat pain threshold were assessed. Patients and controls also rated quality and intensity of the stimuli., Results: The authors found no difference on contact heat evoked potential amplitude (P = 0.59) or latency of N2 or P2 wave (P = 0.62 and P = 0.75, respectively) between patients and controls. In addition, there was no difference in regard to pain intensity scores or pain quality. The patients and controls had similar warmth detection threshold and heat pain threshold. Somatosensory evoked potentials, amplitude, and latency were within normal range and similar for the two groups., Conclusions: The findings suggest that the processing of non-painful and painful stimuli is preserved in patients with mild to moderate Alzheimer's disease., (© 2015 American Academy of Pain Medicine. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The hypoxia-inducible factor (HIF)-1 and β-catenin protective pathways represent the two most significant cellular responses that are activated in response to acute kidney injury. We previously reported that murine mucin (Muc)1 protects kidney function and morphology in a mouse model of ischemia-reperfusion injury (IRI) by stabilizing HIF-1α, enhancing HIF-1 downstream signaling, and thereby preventing metabolic stress (Pastor-Soler et al. Muc1 is protective during kidney ischemia-reperfusion injury. Am J Physiol Renal Physiol 308: F1452-F1462, 2015). We asked if Muc1 regulates the β-catenin protective pathway during IRI as 1) β-catenin nuclear targeting is MUC1 dependent in cultured human cells, 2) β-catenin is found in coimmunoprecipitates with human MUC1 in extracts of both cultured cells and tissues, and 3) MUC1 prevents β-catenin phosphorylation by glycogen synthase kinase (GSK)3β and thereby β-catenin degradation. Using the same mouse model of IRI, we found that levels of active GSK3β were significantly lower in kidneys of control mice compared with Muc1 knockout (KO) mice. Consequently, β-catenin was significantly upregulated at 24 and 72 h of recovery and appeared in the nuclear fraction at 72 h in control mouse kidneys. Both β-catenin induction and nuclear targeting were absent in Muc1 KO mice. We also found downstream induction of β-catenin prosurvival factors (activated Akt, survivin, transcription factor T cell factor 4 (TCF4), and its downstream target cyclin D1) and repression of proapoptotic factors (p53, active Bax, and cleaved caspase-3) in control mouse kidneys that were absent or aberrant in kidneys of Muc1 KO mice. Altogether, the data clearly indicate that Muc1 protection during acute kidney injury proceeds by enhancing both the HIF-1 and β-catenin protective pathways., (Copyright © 2016 the American Physiological Society.)

Background: Following oxaliplatin treatment, acute neurotoxicity symptoms are suggested to be correlated with both the development and degree of chronic neuropathy., Aims: The aim of this clinical commentary was to examine different methods to assess acute cold allodynia and dysesthesia in patients treated with adjuvant oxaliplatin., Methods: Nine patients over the age of 18 years scheduled for standard adjuvant treatment with capecitabine and oxaliplatin were included. Patients were asked to come for two visits: a baseline visit before and a follow-up visit within 5 days after treatment. Patients were examined with questionnaires, thermal tests, and the thermal grill., Results: All patients reported neurotoxicity, and they all had abnormal cold sensitivity. The only significant changes observed were increased ratings of pain, unpleasantness, and pricking sensations to holding a ~8°C metal cylinder for 10 s and an increased intensity of unpleasantness and pricking sensation to the 20-s contact with the 10°C plates of the thermal grill on the palmar hand., Conclusions: he results showed that the palm of the hand is the most sensitive part of the body when detecting oxaliplatin-induced cold allodynia, and the use of a cold metal cylinder seems as a promising sensitive method., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

It remains challenging to produce decisive vaccines against MUC1, a tumor-associated antigen widely expressed by pancreas, breast and other tumors. Employing clinically relevant mouse models, we ruled out such causes as irreversible T-cell tolerance, inadequate avidity, and failure of T-cells to recognize aberrantly glycosylated tumor MUC1. Instead, every tested MUC1 preparation, even non-glycosylated synthetic 9mer peptides, induced interferon gamma-producing CD4(+) and CD8(+) T-cells that recognized glycosylated variants including tumor-associated MUC1. Vaccination with synthetic peptides conferred protection as long as vaccination was repeated post tumor challenge. Failure to revaccinate post challenge was associated with down-regulated tumor MUC1 and MHC molecules. Surprisingly, direct admixture of MUC1-expressing tumor with MUC1-hyperimmune T-cells could not prevent tumor outgrowth or MUC1 immunoediting, whereas ex vivo activation of the hyperimmune T-cells prior to tumor admixture rendered them curative. Therefore, surrogate T-cell preactivation outside the tumor bed, either in culture or by repetitive vaccination, can overcome tumor escape.

De novo design and chemical synthesis of proteins and of other artificial structures that mimic them is a central strategy for understanding protein folding and for accessing proteins with new functions. We have previously described carbohydrates that act as templates for the assembly of artificial proteins, so-called carboproteins. The hypothesis is that the template preorganizes the secondary structure elements and directs the formation of a tertiary structure, thus achieving structural economy in the combination of peptide, linker, and template. We speculate that the structural information from the template could facilitate protein folding. Here we report the design and synthesis of three-helix-bundle carboproteins on deoxyhexopyranosides. The carboproteins were analyzed by CD, analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and NMR spectroscopy, and this revealed the formation of the first compact and folded monomeric carboprotein, distinctly different from a molten globule. En route to this carboprotein we observed a clear effect originating from the template on protein folding., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ischemia-reperfusion injury (IRI) due to hypotension is a common cause of human acute kidney injury (AKI). Hypoxia-inducible transcription factors (HIFs) orchestrate a protective response in renal endothelial and epithelial cells in AKI models. As human mucin 1 (MUC1) is induced by hypoxia and enhances HIF-1 activity in cultured epithelial cells, we asked whether mouse mucin 1 (Muc1) regulates HIF-1 activity in kidney tissue during IRI. Whereas Muc1 was localized on the apical surface of the thick ascending limb, distal convoluted tubule, and collecting duct in the kidneys of sham-treated mice, Muc1 appeared in the cytoplasm and nucleus of all tubular epithelia during IRI. Muc1 was induced during IRI, and Muc1 transcripts and protein were also present in recovering proximal tubule cells. Kidney damage was worse and recovery was blocked during IRI in Muc1 knockout mice compared with congenic control mice. Muc1 knockout mice had reduced levels of HIF-1α, reduced or aberrant induction of HIF-1 target genes involved in the shift of glucose metabolism to glycolysis, and prolonged activation of AMP-activated protein kinase, indicating metabolic stress. Muc1 clearly plays a significant role in enhancing the HIF protective pathway during ischemic insult and recovery in kidney epithelia, providing a new target for developing therapies to treat AKI. Moreover, our data support a role specifically for HIF-1 in epithelial protection of the kidney during IRI as Muc1 is present only in tubule epithelial cells., (Copyright © 2015 the American Physiological Society.)

Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)-binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine-based fluorescent probes in vitro and rationalize our results by electronic structure calculations., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and purpose Conventional neurophysiological techniques do not assess the function of nociceptive pathways and are inadequate to detect abnormalities in patients with small-fiber damage. This overview aims to give an update on the methods and techniques used to assess small fiber (Aδ- and C-fibers) function using evoked potentials in research and clinical settings. Methods Noxious radiant or contact heat allows the recording of heat-evoked brain potentials commonly referred to as laser evoked potentials (LEPs) and contact heat-evoked potentials (CHEPs). Both methods reliably assess the loss of Aδ-fiber function by means of reduced amplitude and increased latency of late responses, whereas other methods have been developed to record ultra-late C-fiber-related potentials. Methodological considerations with the use of LEPs and CHEPs include fixed versus variable stimulation site, application pressure, and attentional factors. While the amplitude of LEPs and CHEPs often correlates with the reported intensity of the stimulation, these factors may also be dissociated. It is suggested that the magnitude of the response may be related to the saliency of the noxious stimulus (the ability of the stimulus to stand out from the background) rather than the pain perception. Results LEPs and CHEPs are increasingly used as objective laboratory tests to assess the pathways mediating thermal pain, but new methods have recently been developed to evaluate other small-fiber pathways. Pain-related electrically evoked potentials with a low-intensity electrical simulation have been proposed as an alternative method to selectively activate Aδ-nociceptors. A new technique using a flat tip mechanical stimulator has been shown to elicit brain potentials following activation of Type I A mechano-heat (AMH) fibers. These pinprick-evoked potentials (PEP) have a morphology resembling those of heat-evoked potentials following activation of Type II AMH fibers, but with a shorter latency. Cool-evoked potentials can be used for recording the non-nociceptive pathways for cooling. At present, the use of cool-evoked potentials is still in the experimental state. Contact thermodes designed to generate steep heat ramps may be programmed differently to generate cool ramps from a baseline of 35◦C down to 32◦C or 30◦C. Small-fiber evoked potentials are valuable tools for assessment of small-fiber function in sensory neuropathy, central nervous system lesion, and for the diagnosis of neuropathic pain. Recent studies suggest that both CHEPs and pinprick-evoked potentials may also be convenient tools to assess sensitization of the nociceptive system. Conclusions In future studies, small-fiber evoked potentials may also be used in studies that aim to understand pain mechanisms including different neuropathic pain phenotypes, such as cold- or touch-evoked allodynia, and to identify predictors of response to pharmacological pain treatment. Implications Future studies are needed for some of the newly developed methods.

A double-headed nucleoside wherein an additional thymine is attached to the 2'-O-position of uridine via a methylene linker is prepared and incorporated into oligonucleotides. With single incorporations of the modified nucleotide monomer, these oligonucleotides form duplexes with the complementary DNA sequences which are thermally less stable as compared to the unmodified duplexes. However, stabilization of bulged duplexes or three way junctions is observed. A cross-strand interaction between two additional thymines is also seen in a DNA-duplex, when specifically introduced in a so-called (+1)-zipper motif, however, much weaker than obtained with the corresponding analogue with the methylene linker directly attached to the 2'-C-position. This demonstrates that the ability to act as a compressed dinucleotide is unique for the latter and due to its perfect preorganization of the additional base in the duplex core.

Purpose: MUC1 is a tumor-associated antigen that is aberrantly expressed in cancer and inflammatory bowel disease (IBD). Even though immune cells express low MUC1 levels, their modulations of MUC1 are important in tumor progression. Consistent with previous clinical data that show increased myeloid-derived suppressor cells (MDSCs) in IBD, we now show that downregulation of MUC1 on hematopoietic cells increases MDSCs in IBD, similar to our data in tumor-bearing mice. We hypothesize that MDSC expansion in IBD is critical for tumor progression., Experimental Design: To mechanistically confirm the linkage between Muc1 downregulation and MDSC expansion, we generated chimeric mice that did not express Muc1 in the hematopoietic compartment (KO→WT). These mice were used in two models of colitis and colitis-associated cancer (CAC) and their responses were compared with wild-type (WT) chimeras (WT→WT)., Results: KO→WT mice show increased levels of MDSCs during colitis and increased protumorigenic signaling in the colon during CAC, resulting in larger colon tumors. RNA and protein analysis show increased upregulation of metalloproteinases, collagenases, defensins, complements, growth factors, cytokines, and chemokines in KO→WT mice as compared with WT→WT mice. Antibody-mediated depletion of MDSCs in mice during colitis reduced colon tumor formation during CAC., Conclusion: Development of CAC is a serious complication of colitis and our data highlight MDSCs as a targetable link between inflammation and cancer. In addition, the lack of MUC1 expression on MDSCs can be a novel marker for MDSCs, given that MDSCs are still not well characterized in human cancers., (©2013 AACR.)

Complex regional pain syndrome (CRPS) is characterised by autonomic, sensory, and motor disturbances. The underlying mechanisms of the autonomic changes in CPRS are unknown. However, it has been postulated that sympathetic inhibition in the acute phase with locally reduced levels of noradrenaline is followed by an up-regulation of alpha-adrenoceptors in chronic CRPS leading to denervation supersensitivity to catecholamines. This exploratory study examined the effect of cutaneous sympathetic activation and inhibition on cutaneous noradrenaline release, vascular reactivity, and pain in CRPS patients and in healthy volunteers. Seven patients and nine controls completed whole-body cooling (sympathetic activation) and heating (sympathetic inhibition) induced by a whole-body thermal suit with simultaneous measurement of the skin temperature, skin blood flow, and release of dermal noradrenaline. CRPS pain and the perceived skin temperature were measured every 5 min during thermal exposure, while noradrenaline was determined from cutaneous microdialysate collected every 20 min throughout the study period. Cooling induced peripheral sympathetic activation in patients and controls with significant increases in dermal noradrenaline, vasoconstriction, and reduction in skin temperature. The main findings were that the noradrenaline response did not differ between patients and controls or between the CRPS hand and the contralateral unaffected hand, suggesting that the evoked noradrenaline release from the cutaneous sympathetic postganglionic fibres is preserved in chronic CRPS patients., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Introduction: We examined the effect of topical lidocaine on the function of small and large fibers in patients with peripheral neuropathic pain due to traumatic or postoperative nerve injury., Methods: In an open-label study, 24 patients were treated with a 5% lidocaine patch for up to 12 weeks. We recorded contact heat evoked potentials (CHEPs) and performed quantitative sensory testing (QST) before and after treatment with the contralateral side as control., Results: Twenty-one patients (mean age 47.6 ± 13.5 years) completed the study. Lidocaine increased cold pain threshold (P = 0.04) and reduced CHEP amplitude (P = 0.007) with no effect on other QST parameters. Patients responding to treatment had less cold detection deficit on the affected side and had a larger increase in cold pain detection threshold following treatment than nonresponders., Conclusions: Controlled trials are warranted to further understand the mechanisms mediating the effects of topical lidocaine., (© 2013 Wiley Periodicals, Inc.)

Additional base pair: The DNA duplex can be extended with an additional Watson-Crick base pair on the same backbone by the use of double-headed nucleotides. These also work as compressed dinucleotides and form two base pairs with cognate nucleobases on the opposite strand., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Brief noxious heat stimuli activate Aδ- and C-fibers and allow contact heat-evoked potentials (CHEPs) to be recorded from the scalp. Under normal conditions, only late responses related to Aδ-fibers can be recorded. This study aimed to demonstrate C-fiber responses to contact heat stimuli. A preferential A-fiber compression blockade of the superficial radial nerve was applied in 22 healthy subjects. Quality and intensity of heat-evoked pain and CHEPs were examined at baseline, during nerve compression, and during nerve compression with simultaneous application of topical capsaicin (5%). During the A-fiber block, three subjects had CHEPs with latencies below 400 ms, eight subjects within 400-800 ms and six subjects (29%) later than 800 ms. Pain intensity to contact heat stimuli after compression was reduced and fewer subjects reported the heat stimuli as stinging. Following nerve compression and capsaicin application, ultralate CHEPs with latencies >800 ms could be recorded in 13 subjects (62%), pain intensity to the contact heat stimuli was increased and the warm/hot-burning pain quality became more intense. The main results of our study are the demonstration of ultralate C-fiber-related CHEPs following A-fiber blockade in 29% of healthy subjects increasing to 62% when the blockade was combined with capsaicin. After blockade of Aδ-fibers we recorded responses with latencies in the range between the latencies of Aδ- and C-fibers suggesting release of Aδ-fibers with slower conduction velocity than normally recorded with CHEPs., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Objective: To examine changes in contact heat evoked potentials (CHEPs) and perceived pain intensity following acute sensitization with topical capsaicin., Methods: CHEPs were recorded before and after 20 min of topical capsaicin application (200 μl, 5%) during skin warming in 22 healthy subjects. To evaluate the sequence effects and skin warming on CHEPs, 10 of these subjects also participated in a control study., Results: Topical capsaicin yielded an increase in contact heat evoked pain ratings (p < 0.0001) and a shortening in N2 latency from a mean 345.2 ± 37.2 ms to 310.2 ± 38.5 ms recorded from the vertex position (p = 0.003, paired t-test). No difference was found in the N2-P2 peak-to-peak amplitude (p = 0.83). These results were unchanged after controlling for sequence effects and skin warming. Following capsaicin, ultralate CHEPs (N2a latencies 970-1352 ms) were recorded in three subjects., Conclusions: Our study showed a decrease in late CHEPs latencies and appearance of ultralate potentials compatible with sensitization of Aδ fibers and C fibers., Significance: Contact heat may be a useful tool to assess sensitization of the pain system., (Copyright © 2011 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.)

We have designed and synthesised a double-headed nucleotide that presents two nucleobases in the interior of a dsDNA duplex. This nucleotide recognises and forms Watson-Crick base pairs with two complementary adenosines in a Watson-Crick framework. Furthermore, with judicious positioning in complementary strands, the nucleotide recognises itself through the formation of a T:T base pair. Thus, two novel nucleic acid motifs can be defined by using our double-headed nucleotide. Both motifs were characterised by UV melting experiments, CD and NMR spectroscopy and molecular dynamics simulations. Both motifs leave the thermostability of the native dsDNA duplex largely unaltered. Molecular dynamics calculations showed that the double-headed nucleotides are accommodated in the dsDNA by entirely local perturbations and that the modified duplexes retain an overall B-type geometry with the dsDNA unwound by around 25 or 60°, respectively, in each of the modified motifs. Both motifs can be accommodated twice in a dsDNA duplex without incurring any loss of stability and extrapolating from this observation and the results of modelling, it is conceivable that both can be multiplied several times within a dsDNA duplex. These new motifs extend the DNA recognition repertoire and may form the basis for a complete series of double-headed nucleotides based on all 16 base combinations of the four natural nucleobases. In addition, both motifs can be used in the design of nanoscale DNA structures in which a specific duplex twist is required., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The mucin MUC1 is typically aberrantly glycosylated by epithelial cancer cells manifested by truncated O-linked saccharides. The resultant glycopeptide epitopes can bind cell surface major histocompatibility complex (MHC) molecules and are susceptible to recognition by cytotoxic T lymphocytes (CTLs), whereas aberrantly glycosylated MUC1 protein on the tumor cell surface can be bound by antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Efforts to elicit CTLs and IgG antibodies against cancer-expressed MUC1 have not been successful when nonglycosylated MUC1 sequences were used for vaccination, probably due to conformational dissimilarities. Immunizations with densely glycosylated MUC1 peptides have also been ineffective due to impaired susceptibility to antigen processing. Given the challenges to immuno-target tumor-associated MUC1, we have identified the minimum requirements to consistently induce CTLs and ADCC-mediating antibodies specific for the tumor form of MUC1 resulting in a therapeutic response in a mouse model of mammary cancer. The vaccine is composed of the immunoadjuvant Pam(3)CysSK(4), a peptide T(helper) epitope and an aberrantly glycosylated MUC1 peptide. Covalent linkage of the three components was essential for maximum efficacy. The vaccine produced CTLs, which recognized both glycosylated and nonglycosylated peptides, whereas a similar nonglycosylated vaccine gave CTLs which recognized only nonglycosylated peptide. Antibodies elicited by the glycosylated tripartite vaccine were significantly more lytic compared with the unglycosylated control. As a result, immunization with the glycosylated tripartite vaccine was superior in tumor prevention. Besides its own aptness as a clinical target, these studies of MUC1 are likely predictive of a covalent linking strategy applicable to many additional tumor-associated antigens.

A series of inhibitors of mammalian 15-lipoxygenase (15-LO) based on a 3,4,5-tri-substituted pyrazole scaffold is described. Replacement of a sulfonamide functionality in the lead series with a sulfamide group resulted in improved physicochemical properties generating analogs with enhanced inhibition in cell-based and whole blood assays., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A series of six double-functionalised nucleosides, in which aromatic moieties were inserted into the 5'-(S)-C-position, were synthesised and incorporated into DNA duplexes. The aromatic moieties were thymine-1-yl, phenyl, 1,2,3-triazol-1-yl, 1,2,3-triazol-4-yl, 4-(uracil-5-yl)-1,2,3-triazol-1-yl and 4-phenyl-1,2,3-triazol-1-yl. The DNA duplexes were studied with UV melting curves, CD spectroscopy and molecular modelling. The results showed that the aromatic moieties in some cases interact in the minor groove forming DNA zipper structures. The strongest specific interaction was found between two thymines or between a thymine and a phenyl group in a crossed (-3)-zipper motif (i.e., with two base pairs interspacing the modifications). Modelling revealed that the interaction is aromatic stacking across the minor groove. Also, the extended uracil-triazole moiety demonstrated zipper contacts in the minor groove as well as binding to the floor of the groove.

3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) inhibitors, more commonly known as statins, represent the gold standard in treating hypercholesterolemia. Although statins are regarded as generally safe, they are known to cause myopathy and, in rare cases, rhabdomyolysis. Statin-dependent effects on plasma lipids are mediated through the inhibition of HMGR in the hepatocyte, whereas evidence suggests that myotoxicity is due to inhibition of HMGR within the myocyte. Thus, an inhibitor with increased selectivity for hepatocytes could potentially result in an improved therapeutic window. Implementation of a strategy that focused on in vitro potency, compound polarity, cell selectivity, and oral absorption, followed by extensive efficacy and safety modeling in guinea pig and rat, resulted in the identification of compound 1b (BMS-644950). Using this discovery pathway, we compared 1b to other marketed statins to demonstrate its outstanding efficacy and safety profile. With the potential to generate an excellent therapeutic window, 1b was advanced into clinical development.

Statins, because of their excellent efficacy and manageable safety profile, represent a key component in the current armamentarium for the treatment of hypercholesterolemia. Nonetheless, myopathy remains a safety concern for this important drug class. Cerivastatin was withdrawn from the market for myotoxicity safety concerns. BMS-423526 [{(3R,5S)-7-[4-(4-fluorophenyl)-6,7-dihydro-2-(1-methylethyl)-5H-benzo[6,7]cyclohepta[1,2-b]pyridin-3-yl]-3,5-dihydroxy-heptenoic acid} sodium salt], similar to cerivastatin in potency and lipophilicity, was terminated in early clinical development due to an unacceptable myotoxicity profile. In this report, we describe the guinea pig as a model of statin-induced cholesterol lowering and myotoxicity and show that this model can distinguish statins with unacceptable myotoxicity profiles from statins with acceptable safety profiles. In our guinea pig model, both cerivastatin and BMS-423526 induced myotoxicity at doses near the ED(50) for total cholesterol (TC) lowering in plasma. In contrast, wide differences between myotoxic and TC-lowering doses were established for the currently marketed, more hydrophilic statins, pravastatin, rosuvastatin, and atorvastatin. This in vivo model compared favorably to an in vitro model, which used statin inhibition of cholesterol synthesis in rat hepatocytes and L6 myoblasts as surrogates of potential efficacy and toxicity, respectively. Our conclusion is that the guinea pig is a useful preclinical in vivo model for demonstrating whether a statin is likely to have an acceptable therapeutic safety margin.

Background: Though the importance of the transmembrane mucin MUC1 in mammary oncogenesis has long been recognized, the relative contributions of the cytoplasmic tail and tandem repeat domains are poorly understood., Methods: To address this, mouse models of mammary carcinogenesis were created expressing full-length, cytoplasmic tail-deleted, or tandem repeat-deleted MUC1 constructs., Results: Overexpression of full-length MUC1 resulted in tumor formation in young mice (≤12 months); however, loss of either the cytoplasmic tail or the tandem repeat domain abrogated this oncogenic capacity. Aged mice in all strains developed late-onset mammary tumors similar to those previously described for the FVB background., Conclusions: This study is the first spontaneous cancer model to address the relative importance of the cytoplasmic tail and tandem repeat domains to MUC1-driven mammary oncogenesis, and suggests that both of these domains are essential for tumor formation.

A bigenic MUC1.Tg/MIN mouse model was developed by crossing Apc/(MIN/+) (MIN) mice with human MUC1 transgenic mice to evaluate MUC1 antigen-specific immunotherapy of intestinal adenomas. The MUC1.Tg/MIN mice developed adenomas at a rate comparable to that of MIN mice and had similar levels of serum MUC1 antigen. A MUC1-based vaccine consisting of MHC class I-restricted MUC1 peptides, a MHC class II-restricted pan-helper peptide, unmethylated CpG oligodeoxynucleotide and GM-CSF caused flattening of adenomas and significantly reduced the number of large adenomas. Immunization was successful in generating a MUC1-directed immune response evidenced by increased MUC1 peptide-specific anti-tumor cytotoxicity and IFN-gamma secretion by lymphocytes.

A series of 2,4,5-tri-substituted imidazoles has proven to be highly potent in inhibiting mammalian 15-lipoxygenase (15-LO) with excellent selectivity over human isozymes 5- and P-12-LO. Non-symmetrical sulfamides (e.g., 21a-n) were found to be suitable replacements for the earlier arylsulfonamide-containing members of this series (e.g., 2, 14a-p). Several members of these series also demonstrated potent inhibition of human 15-LO in a cell-based assay.

The preferred absolute configuration of two series of F(1)F(0)-ATP synthase inhibitors was determined. Although the configuration of the active enantiomer in each series is different, each series presents the same 'triaryl' pharmacophore to the enzyme binding site.

A series of inhibitors of mammalian 15-lipoxygenase based on tryptamine and homotryptamine scaffolds is described. Compounds with aryl substituents at C-2 of the indole core of tryptamine and homotryptamine sulfonamides (e.g., 37a-p) proved to be potent inhibitors of the isolated enzyme. Selected compounds also demonstrated desirable inhibition selectivities over isozymes 5- and P-12-LO.

A series of benzodiazepine-based inhibitors of mitochondrial F(1)F(0) ATP hydrolase were prepared and evaluated for their ability to selectively inhibit the enzyme in the forward direction. Compounds from this series showed excellent potency and selectivity for ATP hydrolase versus ATP synthase, suggesting a potentially beneficial profile useful for the treatment of ischemic heart disease.

A series of substituted guanidine derivatives were prepared and evaluated as potent and selective inhibitors of mitochondrial F(1)F(0) ATP hydrolase. The initial thiourethane derived lead molecules possessed intriguing in vitro pharmacological profiles, though contained moieties considered non-drug-like. Analogue synthesis efforts led to compounds with maintained potency and superior physical properties. Small molecules in this series which potently and selectivity inhibit ATP hydrolase and not ATP synthase may have utility as cardioprotective agents.

A cystic fibrosis (CF) mouse expressing the human mucin MUC1 transgene (CFM) reverted the CF/Muc1(-/-) phenotype (little mucus accumulated in the intestine) to that of CF mice expressing mouse Muc1, which exhibited increased mucus accumulation. Western blots and immunohistochemical analysis showed that the MUC1 protein was markedly increased in CFM mice in which it was both membrane bound and secreted into the intestinal lumen. Studies to determine the reason for increased levels of the extracellular domain of MUC1 mucin identified mRNA and protein of two novel splice variants and the previously described secreted MUC1 lacking the cytoplasmic tail (MUC1/SEC). Novel MUC1 splice variants, CT80 and CT58, were both transmembrane proteins with cytoplasmic tails different from the normal MUC1. The MUC1-CT80 and MUC1/SEC forms are found expressed mainly in the CFM mice intestines. Thus MUC1 expression is increased, and it appears that alternate cytoplasmic tails may change its role in signaling. MUC1 could be an important contributor to the CF intestinal phenotype.

Human mucin 1 (MUC1) is an epithelial mucin glycoprotein that is overexpressed in 90% of all adenocarcinomas including breast, lung, pancreas, prostate, stomach, colon, and ovary. MUC1 is a target for immune intervention, because, in patients with solid adenocarcinomas, low-level cellular and humoral immune responses to MUC1 have been observed, which are not sufficiently strong to eradicate the growing tumor. The hypothesis for this study is that enhancing MUC1-specific immunity will result in antitumor immunity. To test this, the authors have developed a clinically relevant breast cancer model that demonstrates peripheral and central tolerance to MUC1 and develops spontaneous tumors of the mammary gland. In these mice, the authors tested a vaccine formulation comprised of liposomal-MUC1 lipopeptide and human recombinant interleukin-2. Results indicate that when compared with untreated mice, immunized mice develop T cells that express intracellular IFN-gamma, are reactive with MHC class I H-2Db/MUC1 tetramer, and are cytotoxic against MUC1-expressing tumor cells in vitro. The presence of MUC1-specific CTL did not translate into a clinical response as measured by time of tumor onset, tumor burden, and survival. The authors demonstrate that some of the immune-evasion mechanisms used by the tumor cells include downregulation of MHC-class I molecule, expression of TGF-beta2, and decrease in IFN-gamma -expressing effector T cells as tumors progress. Finally, utilizing an injectable breast cancer model, the authors show that targeting a single tumor antigen may not be an effective antitumor treatment, but that immunization with dendritic cells fed with whole tumor lysate is effective in breaking tolerance and protecting mice from subsequent tumor challenge. A physiologically relevant spontaneous breast cancer model has been developed to test improved immunotherapeutic approaches.

Background: Surgical resection of pancreatic tumors removes gross disease but not metastases. Adjuvant therapy such as chemotherapy and radiation treatment is of little value in metastatic pancreatic cancer. The hypothesis of this investigation is that specific and effective immunotherapeutic vaccine (dendritic/tumor cell fusion) will activate cytotoxic T lymphocytes (CTLs), leading to the eradication of spontaneous pancreatic cancer., Methods: We have developed a double transgenic mouse model (MET) that forms spontaneous pancreatic tumors and expresses the human MUC1 antigen. Seven-week-old MET mice (n = 8) were treated every 3 weeks with the vaccine. In addition, these mice received 50 microg of superantigen staphylococcal enterotoxin B (SEB), a known T cell stimulant, prior to the first vaccination. A second treatment group received SEB alone (n = 8) and controls received no treatment (n = 9). MUC1-specific CTLs were measured by chromium release assay. At 10 weeks of age and at necropsy, MUC1 serum levels were measured using a MUC1-specific ELISA., Results: Mice were known to harbor microscopic foci of cancer at birth. Survival was enhanced in vaccine as well as SEB-treated mice (75% CI +/- 0.42) compared to controls (11% CI +/- 0.28) and both groups of treated mice exhibited mature CTLs without in vitro stimulation. MUC1 serum levels of the vaccine group were 50% less than that of control (P < 0.04) at 10 weeks. MUC1 serum levels directly correlated with tumor weight at necropsy (r = 0.86)., Conclusions: This is the first evidence that MUC1-specific CTLs can be stimulated to enhance survival in a spontaneous tumor model.

A 68-year-old woman was admitted for open heart surgery. She had no history of allergy, but had suffered acute heart failure after having received protamine twice. Possible predisposition to adverse reactions is discussed and ways of reducing adverse reactions to protamine are suggested.