Inflammatory pathways confer resistance to chemoradiotherapy in anal squamous cell carcinoma.

Anal squamous cell carcinoma (ASCC) is associated with immunosuppression and infection with human papillomavirus (HPV). Response to standard chemoradiotherapy (CRT) varies considerably. A comprehensive molecular characterization of CRT resistance is lacking, and little is known about the interplay between tumor immune contexture, host immunity, and immunosuppressive and/or immune activating effects of CRT. Patients with localized ASCC, treated with CRT at three different sites of the German Cancer Consortium (DKTK) were included. Patient cohorts for molecular analysis included baseline formalin fixed paraffin embedded biopsies for immunohistochemistry (n = 130), baseline RNA sequencing (n = 98), peripheral blood immune profiling (n = 47), and serum cytokine measurement (n = 35). Gene set enrichment analysis showed that pathways for IFNγ, IFNα, inflammatory response, TNFα signaling via NF-κB, and EMT were significantly enriched in poor responders (all p < 0.001). Expression of interferon-induced transmembrane protein 1 (IFITM1), both on mRNA and protein levels, was associated with reduced Freedom from locoregional failure (FFLF, p = 0.037) and freedom from distant metastasis (FFDM, p = 0.014). An increase of PD-L1 expression on CD4+ T-cells (p < 0.001) and an increase in HLA-DR expression on T-cells (p < 0.001) was observed in the peripheral blood after CRT. Elevated levels of regulatory T-cells and CXCL2 were associated with reduced FFLF (p = 0.0044 and p = 0.004, respectively). Inflammatory pathways in tissue in line with elevated levels of regulatory T-cells and CXCL2 in peripheral blood are associated with resistance to CRT. To counteract this resistance mechanism, the RADIANCE randomized phase-2 trial currently tests the addition of the immune checkpoint inhibitor durvalumab to standard CRT in locally advanced ASCC.

The role of loneliness in the development of depressive symptoms among partnered dementia caregivers: Evidence from the English Longitudinal Study of Aging.

BACKGROUND: Depressive symptoms are highly prevalent among partnered dementia caregivers, but the mechanisms are unclear. This study examined the mediating role of loneliness in the association between dementia and other types of care on subsequent depressive symptoms. METHODS: Prospective data from partnered caregivers were drawn from the English Longitudinal Study of Aging. The sample consisted of 4,672 partnered adults aged 50-70 living in England and Wales, followed up between 2006-2007 and 2014-2015. Caregiving was assessed across waves 3 (2006-2007), 4 (2008-2009), and 5 (2010-2011), loneliness at wave 6 (2012-2013), and subsequent depressive symptoms at wave 7 (2014-15). Multivariable logistic regression models were used to assess the association between caregiving for dementia and depressive symptoms compared to caregiving for other illnesses (e.g., diabetes, coronary heart disease (CHD), cancer, and stroke). Binary mediation analysis was used to estimate the indirect effects of caregiving on depressive symptoms via loneliness. RESULTS: Care for a partner with dementia was associated with higher odds of depressive symptoms at follow-up compared to those not caring for a partner at all (odds ratio [OR] = 2.6, 95% confidence intervals [CI]: 1.4, 5.1). This association was partially mediated by loneliness (34%). Care for a partner with other conditions was also associated with higher odds of depressive symptoms compared to non-caregiving partners (OR = 1.7, 95% CI: 1.2, 2.5), but there was no evidence of an indirect pathway via loneliness. CONCLUSION: Loneliness represents an important contributor to the relationship between dementia caregiving and subsequent depressive symptoms; therefore, interventions to reduce loneliness among partnered dementia caregivers should be considered.

Facile synthesis of one-dimensional organometallic-organic hybrid polymers based on a diphosphorus complex and flexible bipyridyl linkers.

The selective synthesis of a series of new "ladderlike" one-dimensional organometallic-organic hybrid polymers is shown. The polymers are obtained from the reaction of the diphosphorus ligand complex [Cp2Mo2(CO)4(η(2)-P2)] with the copper salt [Cu(CH3CN)4]BF4 in the presence of flexible organic bipyridyl linkers in high selectivity. This unique behaviour is supported by DFT calculations.

Probing Charges on the Atomic Scale by Means of Atomic Force Microscopy.

Kelvin probe force spectroscopy was used to characterize the charge distribution of individual molecules with polar bonds. Whereas this technique represents the charge distribution with moderate resolution for large tip-molecule separations, it fails for short distances. Here, we introduce a novel local force spectroscopy technique which allows one to better disentangle electrostatic from other contributions in the force signal. It enables one to obtain charge-related maps at even closer tip-sample distances, where the lateral resolution is further enhanced. This enhanced resolution allows one to resolve contrast variations along individual polar bonds.

Dynamics of the ion cyclotron resonance effect on amino acids adsorbed at the interfaces.

In this study we show a reproduction of the Zhadin experiment, which consists of the transient increase of the electrolytic current flow across an aqueous solution of L-arginine and L-glutamic acid induced by a proper low frequency alternating magnetic field superimposed to a static magnetic field of higher strength. We have identified the mechanisms that were at the origin of the so-far poor reproducibility of the above effect: the state of polarization of the electrode turned out to be a key parameter. The electrochemical investigation of the system shows that the observed phenomenon involves the transitory activation of the anode due to ion cyclotron frequency effect, followed again by anode passivation due to the adsorption of amino acid and its oxidation products. The likely occurrence of similar ion cyclotron resonance (ICR) phenomena at biological membranes, the implications on ion circulation in living matter, and the consequent biological impact of environmental magnetic fields are eventually discussed.

Efficacy of tacrolimus in rheumatoid arthritis patients who have been treated unsuccessfully with methotrexate: a six-month, double-blind, randomized, dose-ranging study.

OBJECTIVE: To assess the efficacy, safety, and optimal dose of tacrolimus monotherapy in patients with rheumatoid arthritis (RA). METHODS: This phase II, randomized, double-blind, placebo-controlled monotherapy study was set in 12 community sites and 9 university-based sites. Two hundred sixty-eight patients with RA who were resistant to or intolerant of methotrexate (mean dose 15.2 mg/week) and had active disease for at least 6 months (mean tender joint count 28.2, mean erythrocyte sedimentation rate 46.5 mm/hour) were randomized to receive treatment after discontinuation of methotrexate. Those who received at least 1 dose of tacrolimus were analyzed; 141 completed the study. Stable dosages of nonsteroidal antiinflammatory drugs and low-dose prednisone were allowed during treatment. All patients were given 1, 3, or 5 mg of tacrolimus or placebo once daily for 24 weeks. The American College of Rheumatology definition of 20% improvement (ACR20) and the tender and swollen joint counts at the end of treatment were the primary outcomes. RESULTS: ACR20 response rates demonstrated a clear dose response. The ACR20 response was observed in 15.5% of patients receiving placebo (95% confidence interval [95% CI] 7.1-23.9%), 29% of the 1 mg tacrolimus group (95% CI 18.3-39.7%) (P < 0.058); 34.4% of the 3 mg group (95% CI 22.7-46.0%) (P < 0.013), and 50% of the 5 mg group (95% CI 37.8-62.3%) (P < or = 0.001). The tender joint count improved statistically significantly in all tacrolimus groups. The swollen joint count, physical function, and patient-assessed pain improved statistically significantly in the 3 mg and 5 mg groups. The incidence of creatinine elevation > or =40% above baseline levels increased in a dose-dependent manner. Dropout rates were high (41-59%) and were more common for inefficacy in the placebo patients (71.4%), whereas they were more common for toxicity in the high-dose tacrolimus groups (31-33%). Discontinuation for creatinine elevation occurred in the 3 mg (3.1%) and 5 mg (10.9%) tacrolimus groups. CONCLUSION: Tacrolimus improved disease activity in methotrexate-resistant or -intolerant patients with RA. A dose response was observed when efficacy and toxicity were assessed at different doses. The optimal dose of tacrolimus appears to be >1 mg but < or=3 mg daily.

On the "unreasonable" effects of ELF magnetic fields upon a system of ions.

A recent experiment on a physical, nonbiological system of ions at room temperature has proved that microscopic ion currents can be induced by applying simultaneously two parallel magnetic fields, one rather weak static field, (-->)B(0) and one much weaker alternating field, (-->) B(ac),[B(ac) approximately 10(-3) B(0)] whose frequency coincides with the cyclotron frequency v = qB(0)/2pim of the selected ion. As a result, ionic bursts lasting up to 20 s and with amplitude up to 10 nA arise. The much larger exchanges of energy induced by thermal agitation (the "kT-problem") appear to play no role whatsoever. We have analyzed this problem in the framework of coherent quantum electrodynamics, reaching the following conclusions: (a) as has been shown in previous articles, water molecules in the liquid and solute ions are involved in their ground state in coherent ordered configurations; (b) ions are able to move without collisions among themselves in the interstices between water coherence domains; (c) because of coherence, ions can follow classical orbits in the magnetic fields. A full quantitative understanding of the experiments is thus reached.

Activation of protein kinase B/cAkt in hepatocytes is sufficient for the induction of expression of the gene encoding glucokinase.

Inhibitors of signalling pathways were used to dissect the mechanism of insulin action on expression of the gene encoding glucokinase in cultured rat hepatocytes. Wortmannin and LY 294002 completely prevented the insulin-induced increase in glucokinase mRNA seen in unhibited cells, indicating that the phosphoinositide 3-kinase module has a key role. A ligand inducible protein kinase B (PKB, also termed cAkt) fusion protein was expressed by using adenoviral transduction of hepatocytes in primary culture. The PKB activity of this protein was shown to be activated in transduced hepatocytes within 30 min of the addition of 4-hydroxytamoxifen and to stay high for 8 h, as a result of serine phosphorylation at position 473 of PKB. The increase in PKB activity was reflected in the hyperphosphorylation of phosphorylated, heat and acid stable regulated by insulin protein (PHAS-I; also termed 4E-BP1, for eukaryotic initiation factor 4E-binding protein 1), a protein involved in the regulation of translation initiation. These effects were comparable to the insulin-induced activation of endogenous PKB and phosphorylation of PHAS-I in non-transduced hepatocytes. The addition of tamoxifen to transduced hepatocytes resulted in an induction of glucokinase mRNA with kinetics and magnitude similar to those of insulin-induced mRNA accumulation. The effect of tamoxifen depended on stimulated PKB activity because it did not occur in hepatocytes that were transduced with a mutant PKB fusion protein that was refractory to activation with tamoxifen. These results establish that acute activation of PKB is sufficient to produce an insulin-like induction of glucokinase in isolated hepatocytes. Together with the inhibition by phosphoinositide 3-kinase inhibitors, they suggest that the activation of PKB might be critical in mediating the induction of glucokinase by insulin. In addition, experiments showed that PD98059 decreased by half the increase in glucokinase mRNA brought about by insulin, suggesting a contributory role of the mitogen-activated protein kinase cascade.

Regulation of sterol regulatory-element binding protein 1 gene expression in liver: role of insulin and protein kinase B/cAkt.

Insulin stimulates the transcription of the sterol regulatory- element binding protein (SREBP) 1/ADD1 gene in liver. Hepatocytes in primary culture were used to delineate the insulin signalling pathway for induction of SREBP1 gene expression. The inhibitors of phosphoinositide 3-kinase (PI 3-kinase), wortmannin and LY 294002, abolished the insulin-dependent increase in SREBP1 mRNA, whereas the inhibitor of the mitogen- activated protein kinase cascade, PD 98059, was without effect. To investigate the role of protein kinase B (PKB)/cAkt downstream of PI 3-kinase, hepatocytes were transduced with an adenovirus encoding a PKB--oestrogen receptor fusion protein. The PKB activity of this recombinant protein was rapidly activated in hepatocytes challenged with 4-hydroxytamoxifen (OHT), as was endogenous PKB in hepatocytes challenged with insulin. The addition of OHT to transduced hepatocytes resulted in accumulation of SREBP1 mRNA, with a time-course and magnitude similar to the effect of insulin in non-transduced cells. The level of SREBP1 mRNA was not increased by OHT in hepatocytes expressing a mutant form of the recombinant protein whose PKB activity was not activated by OHT. Thus acute activation of PKB is sufficient to induce SREBP1 mRNA accumulation in primary hepatocytes, and might be the major signalling event by which insulin induces SREBP1 gene expression in the liver.

Rapid and sensitive detection of enhanced green fluorescent protein expression in paraffin sections by confocal laser scanning microscopy.

Various forms of green fluorescent protein (GFP) have become important reporters of gene transfer and expression after transfection or infection of cells in cell culture. Frequently, molecular biological assays (Northern blots, PCR) are applied to detect reporter gene expression in target organs. However, these methods are not suitable for evaluation of tissue- or cell-specific expression which would be of great interest especially in case of using tissue-specific promoters. Therefore, organs of transgenic mice with the enhanced green fluorescent protein (EGFP) gene under control of the cytomegalovirus (CMV) promoter were processed for histology by formaldehyde fixation and embedding in paraffin. Sections were deparaffinized, mounted and evaluated for fluorescence in a confocal laser scanning microscope. This method combines the advantages of direct exploitation of tissue sections without further staining procedures with evaluable tissue-, cell-, and even subcellular-specific distribution patterns of EGFP expression in tissues. Results obtained by direct evaluation of EGFP fluorescence in paraffin sections were confirmed by immunohistochemical staining with anti-EGFP. In the present report, we demonstrate that application of confocal microscopy on routinely processed histological preparations is very suitable for determining gene transfer efficiency and promotor activities.

Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b(6)f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b(6)f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b(6)f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.

Stable long-term germ-line transmission of transgene integration sites in mice.

Transgenic mice provide a valuable tool in all fields of basic and applied biological and medical research. In this study, we describe the fate of integrated transgenes in the mammalian host genome over a large number of generations. The stability of the germ-line transmission of integrated tyrosinase transgene copies was monitored up to generation F20 in a large number of individuals from seven transgenic mouse lines. Phenotypic and molecular genetic analysis of the offspring both within the different lines and in cross-breeding experiments revealed the high stability of the transgene integration sites in mice. Only very few individuals were affected by a transgene copy loss. These results indicate that, once homozygous transgenic lines are established, breeding programs can be continued to a high number of generations without further stringent molecular genetic analysis.

Identification of cis-acting RNA leader elements required for chloroplast psbD gene expression in Chlamydomonas.

The psbD mRNA of Chlamydomonas reinhardtii is one of the most abundant chloroplast transcripts and encodes the photosystem II reaction center polypeptide D2. This RNA exists in two forms with 5' untranslated regions of 74 and 47 nucleotides. The shorter form, which is associated with polysomes, is likely to result from processing of the larger RNA. Using site-directed mutagenesis and biolistic transformation, we have identified two major RNA stability determinants within the first 12 nucleotides at the 5' end and near position -30 relative to the AUG initiation codon of psbD. Insertion of a polyguanosine tract at position -60 did not appreciably interfere with translation of psbD mRNA. The same poly(G) insertion in the nac2-26 mutant, which is known to be deficient in psbD mRNA accumulation, stabilized the psbD RNA. However, the shorter psbD RNA did not accumulate, and the other psbD RNAs were not translated. Two other elements were found to affect translation but not RNA stability. The first comprises a highly U-rich sequence (positions -20 to -15), and the second, called PRB1 (positions -14 to -11), is complementary to the 3' end of the 16S rRNA. Changing the PRB1 sequence from GGAG to AAAG had no detectable effect on psbD mRNA translation. However, changing this sequence to CCUC led to a fourfold diminished rate of D2 synthesis and accumulation. When the psbD initiation codon was changed to AUA or AUU, D2 synthesis was no longer detected, and psbD RNA accumulated to wild-type levels. The singular organization of the psbD 5' untranslated region could play an important role in the control of initiation of psbD mRNA translation.

Human nerve growth factor beta (hNGF-beta): mammary gland specific expression and production in transgenic rabbits.

Transgenic rabbits carrying gene constructs encoding human nerve growth factor beta (hNGF-beta) cDNA were generated. Expression of hNGF-beta mRNA was restricted to the mammary gland of lactating rabbits. Western Blot analysis revealed a polypeptide of 13.2 kDa in the milk of transgenic animals. hNGF-beta was purified from the milk by a two-step chromatographic procedure. Electrospray mass spectroscopy analysis of purified hNGF-beta depicted a molecular weight of 13,261 Da per subunit. The biological activity of the hNGF-beta was tested using PC12W2 cells and cultures of dorsal root ganglion neurons from chicken embryos. Crude defatted milk from transgenic animals and purified hNGF-beta demonstrated full biological activity when compared to commercial recombinant hNGF-beta.

The 2.3 kb smooth muscle myosin heavy chain promoter directs gene expression into the vascular system of transgenic mice and rabbits.

BACKGROUND: Smooth muscle cells (SMC) are a preferential target for gene therapeutic approaches in atherosclerosis and restenosis. However, the undesirable expression of putative therapeutic genes in tissues other than the vascular wall is a considerable safety limitation for clinical trials, thus requiring the identification of a smooth-muscle-specific promoter sequence. Since the 2.3 kb rabbit Smooth Muscle Myosin Heavy Chain (SMHC) promoter was shown to be transcriptionally active in primary vascular but not visceral or other non-SMC in vitro, this fragment was chosen for in vivo analysis. METHODS AND RESULTS: Transgenic mice and rabbits were established expressing a luciferase reporter gene under control of the 2.3 kb rabbit SMHC promoter. In contrast to the endogenous expression pattern of the SMHC gene both species revealed light emission predominantly in the arterial system including coronary arteries. Low activities were measured in large veins and the gastrointestinal system. In situ hybridization of murine embryos using a luciferase riboprobe confirmed reporter gene expression in large arteries with no detectable mRNA in the viscera. Unlike adult animals, ectopic luciferase activities were found in ventricular myocardium during murine development ceasing 1 week post partum. CONCLUSIONS: In two animal species, the 2.3 kb SMHC promoter appeared to be effective in discriminating between the pathways regulating vascular and visceral smooth muscle gene expression. The vascular-specific expression profile of the 2.3 kb SMHC promoter suggests that the 2.3 kb SMHC promoter contains the regulatory elements necessary for selective gene targeting into vascular SMC of large arteries including coronary arteries in vivo.

Cardiac specific expression of the green fluorescent protein during early murine embryonic development.

We demonstrate the establishment of transgenic mice, where the expression of the green fluorescent protein (GFP) is under control of the human cardiac alpha-actin promoter. These mice display cardiac specific GFP expression already during early embryonic development. Prominent GFP fluorescence was observed at the earliest stage of the murine heart anlage (E8). Cardiomyocytes of different developmental stages proved GFP positive, but the intensity varied between cells. We further show that contractions of single GFP positive cardiomyocytes can be monitored within the intact embryo. At later stages of embryonic development, the skeletal musculature was also GFP positive, in line with the known expression pattern of cardiac alpha-actin. The tissue specific labeling of organs is a powerful new tool for embryological as well as functional investigations in vivo.

The expression of the C. elegans labial-like Hox gene ceh-13 during early embryogenesis relies on cell fate and on anteroposterior cell polarity.

Clusters of homeobox-containing HOM-C/hox genes determine the morphology of animal body plans and body parts and are thought to mediate positional information. Here, we describe the onset of embryonic expression of ceh-13, the Caenorhabditis elegans orthologue of the Drosophila labial gene, which is the earliest gene of the C. elegans Hox gene cluster to be activated in C. elegans development. At the beginning of gastrulation, ceh-13 is asymmetrically expressed in posterior daughters of anteroposterior divisions, first in the posterior daughter of the intestinal precursor cell E and then in all posterior daughters of the AB descendants ABxxx. In this paper, we present evidence that supports position-independent activation of ceh-13 during early C. elegans embryogenesis, which integrates cell fate determinants and cell polarity cues. Our findings imply that mechanisms other than cell-extrinsic anteroposterior positional signals play an important role in the activation and regulation of the C. elegans Hox gene ceh-13.

Allele-specific suppression of a Saccharomyces cerevisiae prp20 mutation by overexpression of a nuclear serine/threonine protein kinase.

The yeast PRP20 protein is homologous to the RCC1 protein of higher eukaryotes and is required for mRNA export and maintenance of nuclear structure. RCC1/PRP20 act as guanine nucleotide exchange factors for the nuclear Ras-like Ran/GSP1 proteins. In a search for prp20-10 allele-specific high-copy-number suppressors, the KSP1 locus, encoding a serine/threonine protein kinase was isolated. Ksp1p is a nuclear protein that is not essential for vegetative growth of yeast. Inactivation of the kinase activity by a mutation affecting the catalytic center of the Ksp1p eliminated the suppressing activity. Based on the isolation of a protein kinase as a high-copy-number suppressor, the phosphorylation of Prp20p was examined. In vivo labeling experiments showed that Prp20p is a phosphoprotein; however, deletion of the KSP1 kinase did not affect Prp20p phosphorylation.

Long-term interferon-alpha therapy induces autoantibodies against epidermis.

BACKGROUND: While interferon (IFN) is known for its immunoregulatory properties, it has also been shown to induce autoimmune disorders such as lupus erythematosus, hypothyroidism, antiphospholipid syndrome and, recently, bullous pemphigus-like eruptions. OBJECTIVE: The purpose of this study was to determine the percentage of antibodies against epidermis induced by IFN-alpha therapy, as detected by indirect immunofluorescence and Western blotting (WB). METHOD: We have studied the sera of 47 patients treated with low doses of IFN-alpha 2a for malignant melanoma or cutaneous T cell lymphoma for a period of 12 months. These sera were tested by standard IIF and WB. RESULTS: 32% of patient sera were positive after 6 months of treatment. Antibodies against epidermis were most often of the pemphigus type as confirmed by WB. Two types of labeling were noted for anti-basement membrane antibodies: basal cytoplasmic pattern in 4 sera and along the dermal side of the basal keratinocytes in 2 sera. CONCLUSION: This study brings biological arguments to confirm the direct role of IFN-alpha in the development of pemphigus-like eruption, and emphasizes the need for clinical follow-up of patients treated with IFN-alpha over a long period of time.