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.

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.

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.

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.

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.

In vitro induction of basal keratinocyte MY7 antigen expression in cutaneous T-cell lymphoma is associated with response to interferon-alfa therapy.

BACKGROUND: Normal basal cell keratinocytes express an antigen recognized by monoclonal antibody MY7. This expression is lost in cutaneous T-cell lymphoma (CTCL) and may be reinduced under interferon-alfa-2a therapy. We investigated whether similar modulation of MY7 antigen could be obtained in vitro and determined the relationship between in vitro modulation and clinical response. SUBJECTS: We studied MY7 expression by basal cell keratinocytes in reconstituted skin specimens from 10 patients with CTCL and from skin specimens from five control patients and determined its modulation by interferon-alfa-2a and interleukin 1 using the indirect immunofluorescence technique. Concurrently, clinical examination and in vivo immunologic study on cutaneous biopsy specimens were carried out for these 10 patients before and while receiving interferon-alfa-2a therapy. RESULTS: In vitro studies showed that in five control specimens MY7 expression by basal cells was constant without modulation by interferon-alfa-2a or interleukin 1. Two of 10 CTCL specimens spontaneously expressed MY7 antigen while an additional five did so after incubation with interferon-alfa-2a and the last three never did. CONCLUSION: The three patients with CTCL for whom no MY7 expression was observed in reconstituted skin studies were "poor responders" to interferon-alfa-2a therapy. The five patients with CTCL for whom in vitro MY7 expression was induced by interferon-alfa-2a were responders. For the last two patients, results were variable. Thus, in vitro MY7 antigen is expressed in normal basal cell keratinocytes, absent in CTCL basal cell keratinocytes, but can be induced by interferon-alfa-2a. Moreover this in vitro modulation appears to be possibly correlated with interferon-alfa efficacy in vivo.

[Blue rubber bleb nevus]. / Blue rubber bleb naevus.

Blue Rubber Bleb Naevus (BRBN) is characterized by the presence of bluish haemangiomas on the skin and in the digestive tract. We report a new familial case of this rare entity. Analysis of this female patient's pedigree confirmed that BRBN is transmitted as an autosomal dominant trait. In this particular case gastrointestinal haemangiomas appeared over a 4-year period. Although all treatments of this disease have proved disappointing on the skin as well as in the digestive tract, we insist on the need for a regular and prolonged surveillance.

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.

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.

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.

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.

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.