Splicing of scorpion toxin gene BmKK2 in HEK 293T cells.
Using GFP as a reporter gene, splicing of scorpion toxin gene BmKK2 was investigated in cultured HEK 293T cells. The results of RT-PCR and western blotting showed that BmKK2's intron could be recognized and spliced in cultured HEK 293T cells. At the same time, a cryptic splicing site of BmKK2 gene was found at the 91st nucleotide site of the second exon, which is a typical form of alternative splicing. For the first time, alternative splicing would partially explain the diversity of scorpion toxins at the gene level. Moreover, replacing BmKK2's intron with BmP03's intron (an artificial BmKK2-BmP03 mosaic gene) did not affect the intron's recognition and splicing, but increased the expression of the toxin-GFP fusion protein by fluorescence imaging, which indicated that both introns may regulate the expression of toxin-GFP fusion protein. The artificial BmKK2-BmP03 mosaic gene was also spliced into two kinds of mRNA molecules, which showed that sequence of intron was not absolutely conserved. The results suggested that introns of scorpion toxin genes BmKK2 and BmP03 increase the diversity of scorpion toxins and regulate the expression of their genes.


Social Support and the Perception of Geographical Slant.
The visual perception of geographical slant is influenced by physiological resources, such as physical fitness, age, and being physically refreshed. In two studies we tested whether a psychosocial resource, social support, can also affect the visual perception of slants. Participants accompanied by a friend estimated a hill to be less steep when compared to participants who were alone (Study 1). Similarly, participants who thought of a supportive friend during an imagery task saw a hill as less steep than participants who either thought of a neutral person or a disliked person (Study 2). In both studies, the effects of social relationships on visual perception appear to be mediated by relationship quality (i.e., relationship duration, interpersonal closeness, warmth). Artifacts such as mood, social desirability, and social facilitation did not account for these effects. This research demonstrates that an interpersonal phenomenon, social support, can influence visual perception.


POEMS syndrome: follow-up study of a case.
We report here the case of a 20-year-old man with POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, M proteins, skin changes). This rare syndrome followed a 3-year history of a syndrome that mimics a chronic inflammatory demyelinating polyneuropathy (CIDP). Treatment with cyclophosphamide induced regression of the syndrome and improved peripheral nerve conduction.


A method of fitting non-symmetric Gompertz functions for characterising malignant growth.
A basic symmetrically asymptotic Gompertz equation was extended to account for more complex growth and life-table data. This extended Gompertz or non-symmetric asymptotic regression equation was found to be suitable for fitting a large variety of data exhibiting growth and experimentally-induced regression effects expressed in the same curve. The statistical and computational properties of this fitting function are discussed in theory and example.


Enantioselective Rhodium-Catalyzed Cycloisomerization of 1,6-Allenynes to access 5/6-Fused Bicycle[4.3.0]nonadienes.
Transition-metal-catalyzed cycloisomerization of 1,n-allenynes represents a powerful synthetic tool to rapidly assemble complex polycyclic skeletons from simple linear substrates. Nevertheless, there are no reports of the asymmetric version of these reactions. Moreover, most of these reactions proceed through a 6-endo-dig cyclization pathway, which preferentially delivers the distal product (via 5/5 rhodacyclic intermediate) rather than the proximal one (via 6/5 rhodacyclic intermediate). Herein, we report an enantioselective rhodium(I)-catalyzed cycloisomerization of 1,6-allenynes to provide the proximal product 5/6-fused bicycle[4.3.0]nonadienes in good yields and with excellent enantioselectivities. Remarkably, this chemistry works perfectly for 1,6-allenynes having a cyclic substituent within the allene component, thereby affording synthetically formidable tricyclic products with excellent enantioselectivities. Moreover, extensive DFT calculations suggest an uncommon pathway involving 5-exo-dig cycloisomerization, ring-expansion, rate-determining alkene isomerization involving Csp3-H activation, C-C activation of the cyclobutene moiety and finally reductive elimination. Deuterium labeling experiments support the rate-determining step involving the C-H bond activation in this transformation.


Keratinocytes from Gorlin Syndrome-induced pluripotent stem cells are resistant against UV radiation.
Gorlin syndrome (GS) is an autosomal dominant genetic disorder involving Patched 1 (PTCH1) mutations. The PTCH1 is a receptor as well as an inhibitor of hedgehog (Hh) to sequester downstream Hh pathway molecules called Smoothened (SMO). PTCH1 mutations causes a variety of GS conditions including falx calcification, odontogenic keratocytes and basal cell carcinomas (BCC). Because PTCH1 is a major driver gene of sporadic BCC, GS patients are characteristically prone to BCC. In order to elucidate the pathological mechanism of BCC-prone GS patients, we investigated keratinocytes derived from GS patient specific iPS cells (G-OFiPSCs) which were generated and reported previously. We found that keratinocytes derived from G-OFiPSCs (GKCs) have increased expression of Hh target molecules. GKCs were irradiated and those cells showed high resistance to UV induced apoptosis. BCL2, known as anti-apoptotic molecule as well as Hh target, significantly increased in GKCs. Several molecules involved in DNA repair, cell cycle control, senescence, and genotoxic stress such as TP53, BRCA1 and GADD45A increased only in GKCs. GKCs are indicated to be resistant to UV irradiation by upregulating molecules which control DNA repair and genotoxic even under DNA damage caused by UV. The anti-apoptotic properties of GKCs may contribute BCC.


Intermittent hypoxia increases melanoma metastasis to the lung in a mouse model of sleep apnea.
Obstructive sleep apnea (OSA) has recently been associated with an increased risk of cancer incidence and mortality in humans. Experimental data in mice have also shown that intermittent hypoxia similar to that observed in OSA patients enhances tumor growth. The aim of this study was to test the hypothesis that intermittent hypoxia mimicking OSA enhances lung metastasis. A total of 75 C57BL/6J male mice (10-week-old) were subjected to either spontaneous or induced melanoma lung metastasis. Normoxic animals breathed room air and intermittent hypoxic animals were subjected to cycles of 20s of 5% O2 followed by 40s of room air for 6h/day. Spontaneous and induced lung metastases were studied after subcutaneous and intravenous injection of B16F10 melanoma cells, respectively. Compared with normoxia, intermittent hypoxia induced a significant increase in melanoma lung metastasis. These animal model results suggest that intermittent hypoxia could contribute to cancer metastasis in patients with OSA.


Allergen-specific immunotherapy with recombinant grass pollen allergens.
Allergen-specific immunotherapy uses aqueous extracts of natural source materials as a basis for preparations to down regulate the allergic response. Recombinant DNA technology has enabled the cloning of many allergens, thus facilitating investigations aimed at improving efficacy and safety of immunotherapy. To determine the effectiveness of a mixture of 5 recombinant grass pollen allergens in reducing symptoms and need for symptomatic medication in patients allergic to grass pollen. A randomized, double-blind, placebo-controlled study of subcutaneous injection immunotherapy was performed in subjects with allergic rhinoconjunctivitis, with or without asthma. Primary endpoint was a symptom medication score compiled from separate symptom and medication scores. Secondary endpoints included a rhinitis quality of life questionnaire, conjunctival provocation, and specific antibody responses. The symptom medication score showed significant improvements in subjects receiving recombinant allergens as opposed to placebo, with reductions in both symptoms and medication usage. The rhinitis quality of life questionnaire revealed clinically relevant significant improvements in overall assessment and in 5 of 7 separate domains, and conjunctival provocation showed a clear trend in favor of active treatment. All treated subjects developed strong allergen-specific IgG(1) and IgG(4) antibody responses. Some patients were not sensitized to Ph l p 5 but nevertheless developed strong IgG antibody responses to that allergen. A recombinant allergen vaccine can be a effective and safe treatment to ameliorate symptoms of allergic rhinitis. The clinical benefit is associated with modification of the specific immune response with promotion of IgG(4) and reduction of IgE antibodies consistent with the induction of IL-10-producing regulatory T cells.


Anti-autophagic and anti-apoptotic effects of memantine in a SH-SY5Y cell model of Alzheimer's disease via mammalian target of rapamycin-dependent and -independent pathways.
Memantine non-competitively blocks the N-methyl-D-aspartate receptor in order to inhibit beta-amyloid (Aβ) secretion, and has been used to treat moderate-to-severe Alzheimer's disease (AD). However, the mechanisms underlying the role of memantine in the autophagy and apoptosis of neuronal cells in AD, as well as the association between neuronal autophagy and apoptosis have yet to be elucidated. The present study aimed to establish an AD cell model overexpressing the 695‑amino‑acid Swedish mutant of Aβ precursor protein (APP695swe) in order to observe the effects of memantine on the cell viability, autophagy and apoptosis of SH‑SY5Y cells in the AD model, and to investigate the associated underlying mechanisms. A pcDNA3.1‑APP695 plasmid was transfected into the SH‑SY5Y cells. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses demonstrated that the AD cell model was successfully established. MTT assays demonstrated that memantine was able to upregulate neuronal cell survival, and acridine orange staining and flow cytometry demonstrated that memantine (5 µM) was able to inhibit neuronal autophagy and apoptosis. Following neuronal autophagy induction by rapamycin, cell apoptosis rates increased significantly. Further experiments revealed that memantine was able to upregulate the expression of signaling molecules phosphorylated (p)-phosphoinositide 3‑kinase, p‑Akt and p‑mammalian target of rapamycin (mTOR), and also inhibited the phosphorylation of the B‑cell lymphoma 2/Beclin‑1 complex via mitogen‑activated protein kinase 8. In conclusion, the results of the present study demonstrated that in the AD cell model, autophagy was able to promote apoptosis. Memantine exerted anti‑autophagic and anti‑apoptotic functions, and mTOR‑dependent as well as ‑independent autophagic signaling pathways were involved in this process. Therefore, these results of the present study strongly supported the use of memantine as a potential therapeutic strategy for AD treatment.


Antigen-dependent suppression of alloresponses by Foxp3-induced regulatory T cells in transplantation.
Adoptive transfer of polyclonal CD4+CD25+ regulatory T cells (Treg) can tolerize transplantation alloresponses. Treg are activated via their specific TCR, but the antigen specificity of wild-type Treg remains elusive, and therefore controlling potency and duration of Treg activity in the transplantation setting is still not feasible. In this study, we used murine graft-versus-host disease (GVHD) as a model system to show that antigen-specific Treg suppress the response of T effector cells to alloantigens in vitro and prevent GVHD in vivo. The suppressive potential of antigen-specific Treg was much greater than that of polyclonal Treg. To acquire large numbers of antigen-specific Treg, we transduced CD4+CD25- cells with foxp3, and found that these foxp3-induced Treg suppress alloresponses in vitro and prevent GVHD in vivo as effectively as naturally derived CD4+CD25+ Treg. Furthermore, we used an antigen-specific CD4 Th1 clone as a source of foxp3-induced Treg after transduction with foxp3, and found those Treg to effectively prevent GVHD in an antigen-dependent manner. The findings of this study provide a basis for the concept that the onset and potency of the suppression by Treg can be regulated, and suggest a novel approach to enhance the feasibility and effectiveness of inducing tolerance by Treg as an adoptive immunotherapy in transplantation.


Addition of subunit γ, K⁺ ions, and lipid restores the thermal stability of solubilized Na,K-ATPase.
Differential scanning calorimetry (DSC) was applied to ascertain the effect caused by K⁺, Na⁺, ATP, detergent, DPPC, DPPE, and subunit γ on the thermostability of Na,K-ATPase. The enthalpy variation (ΔH) for the thermal denaturation of the membrane-bound is twice the ΔH value obtained for solubilized Na,K-ATPase. Denaturation occurs in five steps for membrane-bound against three steps for the solubilized enzyme, therefore a multi-step unfolding process. In the presence of Na⁺, the melting temperature is 61.6°C, and the ΔH is lower as compared with the ΔH obtained in the presence or in the absence of K⁺. Addition of ATP does not alter the transition temperatures significantly, but the shape of the curve is modified. Subunit γ probably stabilizes Na,K-ATPase in the beginning of thermal unfolding, and different amounts of detergents in the solubilized sample change the protein stability. Reconstitution of Na,K-ATPase into a liposome shows that lipids exert a protector effect. These results reveal differences on the thermostability depending on the conformation of Na,K-ATPase. They are relevant because it allows a comparison with future studies, e.g. how the composition of the membrane interferes on the stability of Na, K-ATPase, elucidating the importance of the lipid type contained in cell membrane.


Comparison of four different mobile devices for measuring heart rate and ECG with respect to aspects of usability and acceptance by older people.
In the area of product design and usability, most products are developed for the mass-market by technically oriented designers and developers for use by persons who themselves are also technically adept by today's standards. The demands of older people are commonly not given sufficient consideration within the early developmental process. In the present study, the usability and acceptability of four different devices meant to be worn for the measurement of heart rate or ECG were analyzed on the basis of qualitative subjective user ratings and structured interviews of twelve older participants. The data suggest that there was a relatively high acceptance concerning these belts by older adults but none of the four harnesses was completely usable. Especially problematic to the point of limiting satisfaction among older subjects were problems encountered while adjusting the length of the belt and/or closing the locking mechanism. The two devices intended for dedicated heart rate recording yielded the highest user ratings for design, and were clearly preferred for extended wearing time. Yet for all the devices participants identified several important deficiencies in their design, as well as suggestions for improvement. We conclude that the creation of an acceptable monitoring device for older persons requires designers and developers to consider the special demands and abilities of the target group.


Age dependent changes in the medial nucleus of the trapezoid body in gerbils.
The results of a quantitative light microscopic analysis of serial glycine immunoreacted sections through the medial nucleus of the trapezoid body (MNTB) of young and old gerbils are presented. Spongiform lesions were prominent in the MNTB of gerbils that were 3 years and older, but were virtually absent in animals below 1 year of age. In old animals the prevalence and density of spongiform lesions were most pronounced in the caudal MNTB and decreased towards the rostral MNTB. Total MNTB volume and rostro-caudal extent were independent of age and the cross-sectional area of MNTB varied in an identical fashion along the MNTB in young and old gerbils. Mean MNTB soma size (cross-sectional area) varied with the age of the animal. In young gerbils soma size increased between 1 and 6 months of age reaching a maximum near 160 microm(2). In old gerbils mean soma size was significantly reduced to 130 microm(2). At all three rostro-caudal positions analyzed along MNTB, soma size varied systematically being largest in the ventro-lateral and smallest in the dorso-medial part of MNTB. The reduction of soma size in old animals appeared uniform across MNTB.


Comparative targeting analysis of KLF1, BCL11A, and HBG1/2 in CD34+ HSPCs by CRISPR/Cas9 for the induction of fetal hemoglobin.
β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB). Imbalanced expression of adult hemoglobin (HbA) induces strong anemia in patients suffering from the disease. However, individuals with natural-occurring mutations in the HBB cluster or related genes, compensate this disparity through γ-globin expression and subsequent fetal hemoglobin (HbF) production. Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). In this study, we thoroughly compared the different CRISPR/Cas9 gene-disruption strategies by gene editing analysis and assessed their safety profile by RNA-seq and GUIDE-seq. All approaches reached therapeutic levels of HbF after gene editing and showed similar gene expression to the control sample, while no significant off-targets were detected by GUIDE-seq. Likewise, all three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices. Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of β-hemoglobinopathies.


Large-scale map of RNA binding protein interactomes across the mRNA life-cycle.
Messenger RNAs (mRNAs) interact with RNA-binding proteins (RBPs) in diverse ribonucleoprotein complexes (RNPs) during distinct life-cycle stages for their processing and maturation. While substantial attention has focused on understanding RNA regulation by assigning proteins, particularly RBPs, to specific RNA substrates, there has been considerably less exploration leveraging protein-protein interaction (PPI) methodologies to identify and study the role of proteins in mRNA life-cycle stages. To address this gap, we generated an RNA-aware RBP-centric PPI map across the mRNA life-cycle by immunopurification (IP-MS) of ~100 endogenous RBPs across the life-cycle in the presence or absence of RNase, augmented by size exclusion chromatography (SEC-MS). Aside from confirming 8,700 known and discovering 20,359 novel interactions between 1125 proteins, we determined that 73% of our IP interactions are regulated by the presence of RNA. Our PPI data enables us to link proteins to life-cycle stage functions, highlighting that nearly half of the proteins participate in at least two distinct stages. We show that one of the most highly interconnected proteins, ERH, engages in multiple RNA processes, including via interactions with nuclear speckles and the mRNA export machinery. We also demonstrate that the spliceosomal protein SNRNP200 participates in distinct stress granule-associated RNPs and occupies different RNA target regions in the cytoplasm during stress. Our comprehensive RBP-focused PPI network is a novel resource for identifying multi-stage RBPs and exploring RBP complexes in RNA maturation.


The interaction between ETS transcription factor family members and microRNAs: A novel approach to cancer therapy.
In cancer biology, ETS transcription factors promote tumorigenesis by mediating transcriptional regulation of numerous genes via the conserved ETS DNA-binding domain. MicroRNAs (miRNAs) act as posttranscriptional regulators to regulate various tumor-promoting or tumor-suppressing factors. Interactions between ETS factors and miRNAs regulate complex tumor-promoting and tumor-suppressing networks. This review discusses the progress of ETS factors and miRNAs in cancer research in detail. We focused on characterizing the interaction of the miRNA/ETS axis with competing endogenous RNAs (ceRNAs) and its regulation in posttranslational modifications (PTMs) and the tumor microenvironment (TME). Finally, we explore the prospect of ETS factors and miRNAs in therapeutic intervention. Generally, interactions between ETS factors and miRNAs provide fresh perspectives into tumorigenesis and development and novel therapeutic approaches for malignant tumors.


Lactic acid bacteria-derived exopolysaccharide: Formation, immunomodulatory ability, health effects, and structure-function relationship.
Exopolysaccharides (EPSs) synthesized by lactic acid bacteria (LAB) have implications for host health and act as food ingredients. Due to the variability of LAB-EPS (lactic acid bacteria-derived exopolysaccharide) gene clusters, especially the glycosyltransferase genes that determine monosaccharide composition, the structure of EPS is very rich. EPSs are synthesized by LAB through the extracellular synthesis pathway and the Wzx/Wzy-dependent pathway. LAB-EPS has a strong immunomodulatory ability. The EPSs produced by different genera of LAB, especially Lactobacillus, Leuconostoc, and Streptococcus, have different immunomodulatory abilities because of their specific structures. LAB-EPS possesses other health effects, including antitumor, antioxidant, intestinal barrier repair, antimicrobial, antiviral, and cholesterol-lowering activities. The bioactivities of LAB-EPS are tightly related to their structures such us monosaccharide composition, glycosidic bonds, and molecular weight (MW). For the excellent physicochemical property, LAB-EPS acts as product improvers in dairy, bakery food, and meat in terms of stability, emulsification, thickening, and gelling. We systematically summarize the detailed process of EPS from synthesis to application, with emphasis on physiological mechanisms of EPS, and specific structure-function relationship, which provides theoretical support for the potential commercial value in the pharmaceutical, chemical, food, and cosmetic industries.


Control of in-vivo differentiation of myeloid leukemic cells--V. Regulation by response to antigen.
Regulation of in-vivo differentiation of myeloid leukemic cells by response to antigen was analysed with different clones of mouse myeloid leukemic cells and human myeloid leukemic cells (HL-60). Differentiation was studied in diffusion chambers implanted into the peritoneal cavity of mice and the antigens used were bovine serum albumin and chicken ovalbumin. It is shown that the presence of either of these antigens in the diffusion chambers can induce differentiation in MGI+D+ mouse and human myeloid leukemic cells, and that pre-immunization with antigen enhanced this in-vivo differentiation. This enhancement showed immunological specificity and was transferred from immunized to non-immunized mice by spleen cells enriched for T lymphocytes. In contrast to these results with MGI+D+ clones of myeloid leukemic cells, clones of WEHI-3B myeloid leukemic cells were induced to differentiate in vivo to the same extent either in the presence or absence of antigen. The results indicate: that in-vivo differentiation of MGI+D+ clones of myeloid leukemic cells can be induced by response to antigen and that in-vivo differentiation of different clones of myeloid leukemic cells can be regulated in different ways.


Relationship of Early Spontaneous Type V Blood Pressure Fluctuation after Thrombolysis in Acute Cerebral Infarction Patients and the Prognosis.
We examined the relationship between an early spontaneous type V blood pressure fluctuation and the post-thrombolysis prognosis of patients with acute cerebral infarction. Patients were admitted consecutively. All patients were categorized into the type V blood pressure fluctuation group or non-type V blood pressure group. Their blood pressure was monitored before thrombolysis and until 6 h after thrombolysis. Baseline data and clinical outcomes were compared. Of 170 patients, 43 (25.2%) had an early type V blood pressure fluctuation. The National Institute of Health Stroke Scale (NIHSS) score before thrombolysis and 24 h after thrombolysis, and the modified Rankin scale score at 90 days differed significantly between the two groups (P < 0.05). Multiple logistic regression analysis showed that an unfavorable prognosis at 3 months was associated with the NIHSS score before thrombolysis (P = 0.000) but probably not with this blood pressure fluctuation (P = 0.058). An early spontaneous type V blood pressure fluctuation is common in patients with acute cerebral infarction who received venous thrombolysis, especially if they have a higher NIHSS score before thrombolysis. The type V blood pressure fluctuation may not influence patients' prognosis; however, this needs to be confirmed in future trials.


Mössbauer spectroscopy and ELISA studies reveal differences between Parkinson's disease and control substantia nigra.
The possible role of iron in the degeneration of nervous cells in Parkinson's disease (PD) was studied with the use of Mössbauer spectroscopy (MS) and enzyme-linked immunoabsorbent assay (ELISA). Mössbauer data were obtained at 90 and 4.1 K from 21 samples of control and 9 samples of parkinsonian substantia nigra (SN). Mössbauer spectra were very similar to those observed in ferritin. Small differences were detected between the spectra obtained from PD and from control SN, and could be due to a slight difference in the composition of the ferritin-like iron cores or due to the presence of about 8% of non-ferritin-like iron in parkinsonian SN. ELISA studies from 11 controls and 6 parkinsonian SN showed a decrease in the concentration of L-chains in wet tissues of PD-SN compared to control SN. The decrease in the amount of L subunits may correspond to a decreased ability of this ferritin to keep iron in a safe form. Iron released from ferritin or neuromelanin (NM) may be the source of such iron, which may cause the difference in the Mössbauer spectra and may trigger oxidative stress leading to cell death.


Loss of heterozygosity of the Mutated in Colorectal Cancer gene is not associated with promoter methylation in non-small cell lung cancer.
'Mutated in Colorectal Cancer' (MCC) is emerging as a multifunctional protein that affects several cellular processes and pathways. Although the MCC gene is rarely mutated in colorectal cancer, it is frequently silenced through promoter methylation. Previous studies have reported loss of heterozygosity (LOH) of the closely linked MCC and APC loci in both colorectal and lung cancers. APC promoter methylation is a marker of poor survival in non-small cell lung cancer (NSCLC). However, MCC methylation has not been previously studied in lung cancer. Therefore, we wanted to determine if MCC is silenced through promoter methylation in lung cancer and whether this methylation is associated with LOH of the MCC locus or methylation of the APC gene. Three polymorphic markers for the APC/MCC locus were analysed for LOH in 64 NSCLC specimens and matching normal tissues. Promoter methylation of both genes was determined using methylation specific PCR in primary tumours. LOH of the three markers was found in 41-49% of the specimens. LOH within the MCC locus was less common in adenocarcinoma (ADC) (29%) than in squamous cell carcinoma (SCC) (72%; P=0.006) or large cell carcinoma (LCC) (75%; P=0.014). However, this LOH was not accompanied by MCC promoter methylation, which was found in only two cancers (3%). In contrast, 39% of the specimens showed APC methylation, which was more common in ADC (58%) than in SCC (13%). Western blotting revealed that MCC was expressed in a subset of lung tissue specimens but there was marked variation between patients rather than between cancer and matching non-cancer tissue specimens. In conclusion, we have shown that promoter methylation of the APC gene does not extend to the neighbouring MCC gene in lung cancer, but LOH is found at both loci. The variable levels of MCC expression were not associated with promoter methylation and may be regulated through other cellular mechanisms.


Nanoliposomal VEGF-R2 peptide vaccine acts as an effective therapeutic vaccine in a murine B16F10 model of melanoma.
The vascular endothelial growth factor receptor-2 (VEGFR-2) plays an important role in melanoma development and progression. Peptide vaccines have shown great potential in cancer immunotherapy by targeting VEGFR-2 as a tumor-associated antigen and boosting the immune response against both tumor cells and tumor endothelial cells. Despite this, the low efficiency of peptide vaccines has resulted in moderate therapeutic results in the majority of studies. Enhancing the delivery of peptide vaccines using nanoliposomes is an important strategy for improving the efficacy of peptide vaccines. In this regard, we designed VEGFR-2-derived peptides restricted to both mouse MHC I and human HLA-A*02:01 using immunoinformatic tools and selected three peptides representing the highest binding affinities. The peptides were encapsulated in nanoliposomal formulations using the film method plus bath sonication and characterized for their colloidal properties. The mean diameter of peptide-encapsulated liposomes was around 135 nm, zeta potential of - 17 mV, and encapsulation efficiency of approximately 70%. Then, vaccine formulations were injected subcutaneously in mice bearing B16F10-established melanoma tumors and their efficiency in triggering immunological, and anti-tumor responses was evaluated. Our results represented that one of our designed VEGFR-2 peptide nanoliposomal formulations (Lip-V1) substantially activated CD4+ (p < 0.0001) and CD8+ (P < 0.001) T cell responses and significantly boosted the production of IFN-γ (P < 0.0001) and IL-4 (P < 0.0001). Furthermore, this formulation led to a significant decrease in tumor volume (P < 0.0001) and enhanced survival (P < 0.05) in mice. Our findings suggest that the nanoliposomal formulation containing VEGFR-2 peptides could be a promising therapeutic vaccination approach capable of eliciting strong antigen-specific immunologic and anti-tumor responses. The online version contains supplementary material available at 10.1186/s12645-023-00213-7.


Differential regulation of the InsP₃ receptor type-1 and -2 single channel properties by InsP₃, Ca²⁺ and ATP.
An elevation of intracellular Ca2+ levels as a result of InsP3 receptor (InsP3R) activity represents a ubiquitous signalling pathway controlling a wide variety of cellular events. InsP3R activity is tightly controlled by the levels of the primary ligands, InsP3, Ca2+ and ATP. Importantly, InsP3Rs are regulated by Ca2+ i in a biphasic manner. Ca2+ release through all InsP3R family members is also modulated dramatically by ATP, albeit with sub-type-specific properties. To ascertain if a common mechanism can account for ATP and Ca2+ regulation of these InsP3R family members, we examined the effects of [ATP] on the Ca2+ dependency of rat InsP3R-1 (rInsP3R-1) and mouse InsP3R-2 (mInsP3R-2) activity expressed in DT40-3KO cells. We used the on-nucleus patch clamp recording technique with various [ATP], [InsP3] and [Ca2+] in the patch pipette and measured single InsP3R channel activity in stably transfected DT40 cells. Under identical conditions, at saturating [InsP3] and [ATP], the activity of rInsP3R-1 and mInsP3R-2 was essentially identical in terms of single channel conductance, maximal achievable open probability (Po) and the [Ca2+] required for activation and inhibition of activity. However, in contrast to rInsP3R-1 at saturating [InsP3], the activity of mInsP3R-2 was unaffected by [ATP]. At lower [InsP3], ATP had dramatic effects on mInsP3R-2 Po, but unlike the rInsP3R-1, this did not occur by altering the relative Ca2+ dependency, but by simply increasing the maximally achievable Po at a particular [InsP3] and [Ca2+]. [InsP3] did not alter the biphasic regulation of activity by Ca2+ in either rInsP3R-1 or mInsP3R-2. Analysis of the single channel kinetics indicated that Ca2+ and ATP modulate the Po predominately by facilitating extended bursting activity of the channel but the underlying biophysical mechanism appears to be distinct for each receptor. Subtype-specific regulation of InsP3R channel activity probably contributes to the fidelity of Ca2+ signalling in cells expressing these receptor subtypes.


Rare-Earth hydroxide nanosheets based ratio fluorescence nanoprobe for dipicolinic acid detection.
We demonstrate a novel ratio fluorescence nanoprobe for dipicolinic acid (DPA) as an anthrax biomarker based on layered rare-earth hydroxide (LRH). 3-Amino-benzenesulfonic acid (AS) was intercalated into layered terbium hydroxide to form composite and then delaminated into nanosheets in formamide. The monolayer nanosheets were beneficial to expose the Ln3+ luminescence centers to the environment more completely, contributing a high sensitive detection to the environment. With the increase of DPA concentration, the emission intensity of AS kept constant which worked as a stable internal reference, while the fluorescence of Tb3+ was enhanced obviously due to the antenna effect. In the 0.05-5.0 μM concentration range, the I544/I360 fluorescence ratio changed with the DPA concentration, which exhibited a good linear relationship (R2 = 0.999) and an ultralow detection limit of 3.8 nM. In addition, the probe showed high selectivity and sensitivity to the DPA detection as an anthrax biomarker, which can be applied in real tap water with good performances. This work could extend the applications of LRH nanosheets in detection and offer an extremely effective and easy technique for detecting DPA.


Vitreous leptin levels in retinal disease.
The purpose of this study was to investigate the relationship between vitreous leptin levels and retinal diseases. Levels of vitreous leptin were evaluated in proliferative diabetic retinopathy (PDR) and a variety of other retinopathies including: macular disease, neovascular maculopathies, primary retinal detachments, and vascular occlusive disease. In patients with PDR (N=7), the average vitreous level of leptin (37.4 ng/ml) was significantly higher than that in patients with PVR (<1.0 ng/ml, P<0.05). Vitreous leptin level in patients with PVR or macular disease (N=18), with or without diabetes, was not significantly different from the control subjects who had retinal detachment only (N=7). The results show that the leptin level in vitreous taps is elevated in PDR. We suggest that leptin plays an active role in PDR.


Derotational distal femur osteotomy combined with medial patellofemoral ligament reconstruction yields satisfactory results in recurrent patellar dislocation with excessive femoral anteversion angle and trochlear dysplasia.
The purpose of this study was to evaluate the clinical outcomes of medial patellofemoral ligament (MPFL) reconstruction combined with derotational distal femur osteotomy in patients with recurrent patellar dislocation who had excessive femoral anteversion angle and trochlear dysplasia. Between 2015 and 2020, 64 patients (64 knees) with recurrent patellar dislocation, who had excessive femoral anteversion angle (≥ 25°) and trochlear dysplasia and were surgically treated using derotational distal femur osteotomy and MPFL reconstruction, were eligible for this retrospective study. These patients were assigned to two groups according to the grade of trochlear dysplasia. Group A (type A trochlear dysplasia, n = 33) and Group B (type B, C, D trochlear dysplasia, n = 31). Preoperative and postoperative patellar tilt angle (PTA), Caton-Deschamps index (CD-I), tibial tubercle-trochlear groove (TT-TG) distance and femoral anteversion angle were evaluated. Patient outcomes were assessed using the preoperative and postoperative International Knee Documentation Committee (IKDC) score, Kujala score, Lysholm score, Tegner score, and visual analog scale (VAS) score. A total of 64 patients (64 knees) were evaluated in this study, with a mean follow-up period of 28.4 ± 3.6 months. There were no cases of wound infection, osteotomy site fractures, deep venous thrombosis of the lower extremities, or re-dislocation in the two groups during the postoperative follow-up period. All patients returned to full extension and flexion. The postoperative Tegner score, Lysholm score, Kujala score, IKDC score, VAS score, PTA, CD-I, TT-TG distance, and femoral anteversion angle were significantly improved compared with the preoperative status (P < 0.05). There was no significant difference between the two groups (n.s.). MPFL reconstruction combined with derotational distal femur osteotomy showed satisfactory clinical outcomes during follow-up in patients with recurrent patellar dislocation who had excessive femoral anteversion angle and trochlear dysplasia. Even patients with high-grade trochlear dysplasia showed satisfactory results. For those patients, additional surgery is not necessary. Level III.


Environmental and childhood lead contamination in the proximity of boat-repair yards in southern Thailand--I: pattern and factors related to soil and household dust lead levels.
High blood lead levels have recently been documented in schoolchildren living in communities adjacent to boat-repair yards in southern Thailand. In this study, the spatial pattern of lead contamination of soil and household dust in an area surrounding several boat-repair yards is described, and household factors associated with elevated dust lead are identified. A cross-sectional spatial study was conducted in a coastal residential area within a distance of 2 km from three major boat-repair yards situated on the east coast of peninsular Thailand. Household dust specimens were collected from an undisturbed position in the residences of children, aged 4-14 years, sampled randomly from all children living in the study area. Soil specimens were obtained from the interstices of a square grid, 70 x 70 m2, superimposed on the area. Geographic coordinates of residence and soil sampling positions were recorded and semivariograms and kriging used to contour the spatial distribution of lead in dust and soil. Environmental lead levels were also modeled in terms of direction and minimum distance from a boat-repair yard and, for household dust lead content, in terms of household variables, including occupation of household members in boat-repair work, type of house construction, and general cleanliness. Household dust and soil lead content ranged from 10 to 3025 mg/kg and from 1 to 7700 mg/kg, respectively. The distribution of soil lead peaked at the location of the boat-repair yards, but outside the yards the distribution was generally below 400 mg/kg and irregular. About 24% of household dust lead specimens were equal to or above 400 mg/kg, but showed significant decrease with increasing distance from the boat-repair yards, at rates of between 7% and 14% per 100 m. In houses where a family member was a worker in one of the major boatyards and in houses where occasional repair of small boats was undertaken, household dust lead levels were significantly elevated, by 65% (95% CI: 18-130%) and 31% (95% CI: 5-63%), respectively. Siting of boat-repair yards at a distance from residential areas and measures to reduce the spread of lead-containing dust are recommended to alleviate the problem of elevated household dust lead levels.


Sex differences in neuropsychiatric symptoms in Alzheimer's disease dementia: a meta-analysis.
Neuropsychiatric symptoms (NPS) are common in individuals with Alzheimer's disease (AD) dementia, but substantial heterogeneity exists in the manifestation of NPS. Sex differences may explain this clinical variability. We aimed to investigate the sex differences in the prevalence and severity of NPS in AD dementia. Literature searches were conducted in Embase, MEDLINE/PubMed, Web of Science Core Collection, Cochrane Central Register of Controlled Trials, PsycINFO, and Google Scholar from inception to February 2021. Study selection, data extraction, and quality assessment were conducted in duplicate. Effect sizes were calculated as odds ratios (OR) for NPS prevalence and Hedges' g for NPS severity. Data were pooled using random-effects models. Sources of heterogeneity were examined using meta-regression analyses. Sixty-two studies were eligible representing 21,554 patients (61.2% females). The majority of the included studies had an overall rating of fair quality (71.0%), with ten studies of good quality (16.1%) and eight studies of poor quality (12.9%). There was no sex difference in the presence of any NPS (k = 4, OR = 1.35 [95% confidence interval 0.78, 2.35]) and overall NPS severity (k = 13, g = 0.04 [- 0.04, 0.12]). Regarding specific symptoms, female sex was associated with more prevalent depressive symptoms (k = 20, OR = 1.60 [1.28, 1.98]), psychotic symptoms (general psychosis k = 4, OR = 1.62 [1.12, 2.33]; delusions k = 12, OR = 1.56 [1.28, 1.89]), and aberrant motor behavior (k = 6, OR = 1.47 [1.09, 1.98]). In addition, female sex was related to more severe depressive symptoms (k = 16, g = 0.24 [0.14, 0.34]), delusions (k = 10, g = 0.19 [0.04, 0.34]), and aberrant motor behavior (k = 9, g = 0.17 [0.08, 0.26]), while apathy was more severe among males compared to females (k = 11, g = - 0.10 [- 0.18, - 0.01]). There was no association between sex and the prevalence and severity of agitation, anxiety, disinhibition, eating behavior, euphoria, hallucinations, irritability, and sleep disturbances. Meta-regression analyses revealed no consistent association between the effect sizes across studies and method of NPS assessment and demographic and clinical characteristics. Female sex was associated with a higher prevalence and greater severity of several specific NPS, while male sex was associated with more severe apathy. While more research is needed into factors underlying these sex differences, our findings may guide tailored treatment approaches of NPS in AD dementia.


Architecture of polymorphisms in the human genome reveals functionally important and positively selected variants in immune response and drug transporter genes.
Genetic polymorphisms can contribute to phenotypic differences amongst individuals, including disease risk and drug response. Characterization of genetic polymorphisms that modulate gene expression and/or protein function may facilitate the identification of the causal variants. Here, we present the architecture of genetic polymorphisms in the human genome focusing on those predicted to be potentially functional/under natural selection and the pathways that they reside. In the human genome, polymorphisms that directly affect protein sequences and potentially affect function are the most constrained variants with the lowest single-nucleotide variant (SNV) density, least population differentiation and most significant enrichment of rare alleles. SNVs which potentially alter various regulatory sites, e.g. splicing regulatory elements, are also generally under negative selection. Interestingly, genes that regulate the expression of transcription/splicing factors and histones are conserved as a higher proportion of these genes is non-polymorphic, contain ultra-conserved elements (UCEs) and/or has no non-synonymous SNVs (nsSNVs)/coding INDELs. On the other hand, major histocompatibility complex (MHC) genes are the most polymorphic with SNVs potentially affecting the binding of transcription/splicing factors and microRNAs (miRNA) exhibiting recent positive selection (RPS). The drug transporter genes carry the most number of potentially deleterious nsSNVs and exhibit signatures of RPS and/or population differentiation. These observations suggest that genes that interact with the environment are highly polymorphic and targeted by RPS. In conclusion, selective constraints are observed in coding regions, master regulator genes, and potentially functional SNVs. In contrast, genes that modulate response to the environment are highly polymorphic and under positive selection.


Frontiers and techniques in plant gene regulation.
Understanding plant gene regulation has been a priority for generations of plant scientists. However, due to its complex nature, the regulatory code governing plant gene expression has yet to be deciphered comprehensively. Recently developed methods-often relying on next-generation sequencing technology and state-of-the-art computational approaches-have started to further our understanding of the gene regulatory logic used by plants. In this review, we discuss these methods and the insights into the regulatory code of plants that they can yield.


Interrelationship between the zeta potential and viscoelastic properties in coacervates complexes.
The formation of the complex coacervate (CC) phases between gum Arabic (GA) and low molecular weight chitosan (Ch) and the interrelationship between the zeta-potential and viscoelastic properties of the coacervate phase were investigated. The maximum charge difference of biopolymers stock dispersion was displayed in a range of pH between 4.0 and 5.5. Titration experiment between the oppositely charged biopolymers showed that the isoelectric point was found at a biopolymers mass ratio (R[GA:Ch]) of R[5.5:1]. Turbidity, size and ζ-potential of the soluble complexes (SC) showed an interrelation with the complex coacervate yield (CCY). Higher CCY values (82.2-88.1%) were obtained in the range from R[3:1] to R[5.5:1]. Change the R[GA:Ch] in dispersion, make possible to produce CC's phases exhibiting cationic (R[1:1] and R[3:1]), neutral (R[5.5:1]) or anionic (R[9:1] and R[7:1]) charged. All CC's exhibited liquid-viscoelastic behavior at lower frequencies and a crossover between G″ and G' at higher frequencies.


Electrogenic reactions and dielectric properties of photosystem II.
This review is focused on the mechanism of photovoltage generation involving the photosystem II turnover. This large integral membrane enzyme catalyzes the light-driven oxidation of water and reduction of plastoquinone. The data discussed in this work show that there are four main electrogenic steps in native complexes: (i) light-induced charge separation between special pair chlorophylls P(680) and primary quinone acceptor Q(A); (ii) P(680)(+) reduction by the redox-active tyrosine Y(Z) of polypeptide D1; (iii) oxidation of Mn cluster by Y(Z)(ox) followed by proton release, and (iv) protonation of double reduced secondary quinone acceptor Q(B). The electrogenicity related to (i) proton-coupled electron transfer between Q(A)(-) and preoxidized non-heme iron (Fe(3+)) in native and (ii) electron transfer between protein-water boundary and Y(Z)(ox) in the presence of redox-dye(s) in Mn-depleted samples, respectively, were also considered. Evaluation of the dielectric properties using the electrometric data and the polarity profiles of reaction center from purple bacteria Blastochloris viridis and photosystem II are presented. The knowledge of the profile of dielectric permittivity along the photosynthetic reaction center is important for understanding of the mechanism of electron transfer between redox cofactors.


Amniotic membrane in the surgical management of acute toxic epidermal necrolysis.
To report a new surgical technique to manage severe acute toxic epidermal necrolysis. Two interventional case reports. Two patients. Case 1: A 6-year-old boy had severe toxic epidermal necrolysis develop after being treated with trimethoprim and sulfamethoxazole for chronic otitis media. Both eyes and eyelids were affected. He underwent bilateral lysis of symblepharon and all adhesions and bilateral amniotic membrane transplantation to the entire ocular surface except the cornea. Loss of eyelid skin required transplantation of amniotic membrane to all four eyelids and strips of amniotic membrane at the eyelid margins. Case 2: An 8-year-old girl with severe toxic epidermal necrolysis associated with mycoplasma pneumonia had bilateral, diffuse keratoconjunctivitis, diffuse corneal epithelial defects, and bilateral symblepharon. Amniotic membrane transplantation was performed bilaterally, using a symblepharon ring in the left eye. Amniotic membrane transplantation. Preservation of normal ocular and eyelid surfaces and prevention of blindness. Case 1: Thirty-six months after bilateral ocular surgery, there is no symblepharon, good ocular surface wetting, and an uncorrected bilateral vision of 20/20. Case 2: Amniotic membrane transplantation protected both ocular surfaces and prevented conjunctival contracture without adhesion of the eyelids to the ocular surface. The central vision was preserved. There was minimal peripheral corneal vascularization and mild conjunctival scarring of the tarsal conjunctival surface 34 months postoperatively. These are the first cases of acute toxic epidermal necrolysis treated with amniotic membrane transplantation and the first use of the procedure on external eyelid surfaces with good healing of the eyelids. This new treatment for acute toxic epidermal necrolysis preserves normal ocular and eyelid surfaces and may prevent blindness.


Real-world effectiveness and safety of vedolizumab induction therapy for ulcerative colitis: A prospective nationwide Polish observational study.
Vedolizumab is recommended as a first-line biological treatment, along with other biological drugs, in ulcerative colitis (UC) patients in whom conventional therapy failed and as a second-line biological treatment following a failure of a tumor necrosis factor alpha (TNF-α) antagonist. We aimed to assess the real-world effectiveness and safety of vedolizumab induction therapy in UC patients treated in the scope of the National Drug Program (NDP) in Poland. The endpoints were the proportions of patients who reached clinical response, clinical remission and mucosal healing at week 14. Partial Mayo scores, Mayo subscores and C-reactive protein (CRP) levels were also evaluated. Our study population consisted of 100 patients (55 biologic-naïve and 45 biologic-exposed). The median total Mayo score at baseline was 10 (interquartile range (IQR): 9-11), and 52 patients (52%) had extensive colitis. The clinical response at week 14 was achieved in 83 (83%) and clinical remission in 24 (24%) cases. Mucosal healing was observed in 56 (62%) patients at week 14. In patients with prior failure of biologic treatment (n = 25), 17 (68%) responded to vedolizumab treatment. A decrease in the median CRP level (from 3.7 mg/L to 2.6 mg/L) and the median total Mayo score (from 10 to 4) was observed. No new safety concerns were recorded and no patients discontinued the treatment due to adverse events (AEs). Vedolizumab was effective and safe as induction therapy for UC in a Polish real-world population including patients with severely active UC and a low number of patients with prior biological treatment failures.


A linear-dendritic cationic vector for efficient DNA grasp and delivery.
This paper presents an attempt to design an efficient and biocompatible cationic gene vector via structural optimization that favors the efficient utilization of amine groups for DNA condensation. To this end, a linear-dendritic block copolymer of methoxyl-poly(ethylene glycol)-dendritic polyglycerol-graft-tris(2-aminoethyl)amine (mPEG-DPG-g-TAEA) was prepared with specially designed multiple functions including strong DNA affinity, endosomal buffering and expected serum-tolerance. Based on the transfection in serum-free and serum-conditioned media, the influences of the polymer structures including the degree of polymerization of DPG and TAEA substitution degree were explored. As compared to polyethylenimine (M(w)=5 kDa) (PEI5k) with similar molecular weight and higher amine density, mPEG-DPG-g-TAEA displayed comparably high DNA affinity due to the special linear-dendritic architecture. Consequently, at very low N/P ratio, mPEG-DPG-g-TAEA vectors could mediate efficient in vitro luciferase expression at levels that are comparable with or even superior to the commercially available Lipofectamine™ 2000, while being apparently higher than PEI5k. The designed vectors exhibit considerably higher cell biocompatibility and better resistance against bovine serum albumin adsorption than PEI5k. The stability of the complexes on coincubation with heparin was found to be largely dependent on the polymer structure. As concluded from the comparative transfection study in the absence/presence of chloroquine, it is likely that the polycation itself could produce endosomal buffering. This linear-dendritic vector shows promising potential for the application of gene delivery.


Towards the cortical representation of form and motion stimuli generated by a retina implant.
A retina implant for restoring basic visual perception in patients who are blind due to photoreceptor loss should not only evoke focal phosphenes at high resolution, but should also generate cortical representations of form and motion. We are currently exploring these potential capabilities in anaesthetised cats. Fibre electrodes were inserted through a small scleral incision onto the retinal surface for stimulation. For the recording of cortical population activities we placed up to 16 fibre electrodes in areas 17 and/or 18. Retinal and cortical electrodes were adjusted to corresponding sites, i.e., overlapping receptive fields. Electrical stimuli were charge-balanced impulses (200 micros, 10-100 microA). Basic form stimuli were generated by the selective and synchronous activation of some of the seven retinal stimulation electrodes. Movement stimuli were generated by spatio-temporal shifting of form stimuli. From multiple microelectrode recordings we computed stimulus-related spatio-temporal cortical activation profiles. We used these profiles to estimate the relations between stimulation distance and spatial resolution (form) and between stimulus velocity and spatio-temporal resolution (movement). Influences by the retino-cortical pathway were assessed by comparing cortical activations evoked by true form or motion stimuli with synthetic responses composed by superpositioning of responses to appropriate subsets of form and motion stimuli. In addition, we compared cortical responses to form and motion stimuli by a receptive-field-based backprojection of cortical activities. We confirmed our previous finding that electrical retina stimulation may yield a spatial resolution of 1-5 degrees visual angle and a temporal resolution of about 20 ms. We found that the spatio-temporal cortical activation profiles are commonly related to retinal form and motion stimuli. Cortical activity analyses showed that for two-point form stimuli the neuronal interaction depends on the stimulation electrodes' distance and that local cortical group activities can exhibit some tuning to the directions or the velocities of moving electrical bars'. Projections of cortical activations to visual space were consistent with electrical form and motion stimulation of the retina. Our data indicate that retinal stimulation with electrical form and motion stimuli can lead to spatio-temporally related cortical activations. However, the selective activation of single cortical neurones with specific visual tuning properties by electrical retina stimulation and the potential adaptation of the visual system to long-term stimulation with retina implants should be addressed in future work.


Mannan binding lectin and the sudden infant death syndrome.
Mannan binding lectin (MBL) may be important for innate immunity and some cases of sudden infant death syndrome (SIDS) may be preceded by bacterial infection. Therefore, relative MBL deficiency might be associated with susceptibility to SIDS. We measured MBL concentrations in 46 SIDS infants and 26 controls. The proportion of subjects with low MBL values was similar in the two groups. However, the mean for the SIDS group (3 micrograms/ml) was higher than that of the controls (2.2 micrograms/ml; P < 0.05). We interpret this difference as due to acute phase responses and suggest these findings are consistent with the view that some cot deaths are preceded by bacterial infections.


Intraspecific variation in the response to CO2 enrichment in seeds and seedlings of Plantago lanceolata L.
Four genotypes of P. lanceolata were grown to maturity at combinations of two levels of atmospheric CO2 concentrations and two temperature conditions. Seed weight was determined, and seed germination and seedling growth were measured for the progeny of each genotype under the same environmental conditions. Overall, high CO2 levels decreased seed weight, increased germination percentage and rate, and increased seedling size. Families differed in their response to CO2 enrichment, and to combinations of CO2 and temperature levels for several characters. These results suggest the existence of genetic variability in P. lanceolata in response to CO2 enrichment.


Bisacodyl removal from contaminated solution by synthesized mesoporous silica using experimental design method.
Most of the medicinal compounds are entered no change into municipal wastewater and more than 90% of it remains in the wastewater. Mesoporous silica is known as thermally stable materials with controllable porosity and morphology. It is specified that these materials possess external and internal surfaces that can be selectively adsorbed the various compounds. In the present study, the synthesized mesoporous silica was studied to remove bisacodyl from polluted solutions. Mesoporous silica was synthesized by simple chemical method from tetraethylorthosilicate, ethanol (C2H5OH) and deionized water at 70 °C temperature. Characteristic of synthesized mesoporous silica was investigated by scanning electron microscopy. The effective parameters such as contact time, pH, adsorbate dose, and initial bisacodyl concentration were optimized for bisacodyl removal using 27 batch adsorption experiments according to design of experiment. The residual bisacodyl concentration was measured by UV-Vis spectrometer at the maximum wavelength of 580 nm. The statistical test and linear regression model were used by MINITAB 16 software for investigation of the main and interaction effects of each factor in the adsorption process. The ANOVA result showed 84% of bisacodyl was removed by synthesized mesoporous silica at optimum condition. The result of regression (R2 > 0.99) indicated that the adsorption process followed the Langmuir isotherm and second order kinetic at optimum conditions. The mesoporous silica is an efficient adsorbent for removing of bisacodyl from the polluted solutions so that it can be used for refining wastewaters containing medical compounds such as bisacodyl.


Statistical methods in handling placebo effect.
A critical issue facing the therapeutic area of neurological diseases is the large number of failed randomized clinical trials, especially when moving from promising Phase 2 trials to failed Phase 3 trials. A common cited reason for these failures is a high placebo response rate that thereby reduces the observed treatment effect. Explanations for this higher than anticipated placebo response include small sample sizes, inadequate study designs and/or analytic methods, baseline characteristics of the trial sample, possible investigator bias and a participant's own expectations and conditional learning. Several innovative study designs and new methodological approaches to statistical analyses have been proposed to handle placebo effects anticipated or observed in double blind, randomized clinical trials (RCT's). This chapter examines current study designs being used to reduce the observed placebo response and statistical analysis methods being employed for addressing this problem in neuroscience clinical trials.


Gintonin attenuates depressive-like behaviors associated with alcohol withdrawal in mice.
Panax ginseng Meyer extracts have been used to improve mood and alleviate symptoms of depression. However, little is known about the extracts' active ingredients and the molecular mechanisms underlying their reported anti-depressive effects. Gintonin is an exogenous lysophosphatidic acid (LPA) receptor ligand isolated from P. ginseng. BON cells, an enterochromaffin cell line, and C57BL/6 mice were used to investigate whether gintonin stimulates serotonin release. Furthermore, the effects of gintonin on depressive-like behaviors following alcohol withdrawal were evaluated using the forced swim and tail suspension tests. Treatment of BON cells with gintonin induced a transient increase in the intracellular calcium concentration and serotonin release in a concentration- and time-dependent manner via the LPA receptor signaling pathway. Oral administration of the gintonin-enriched fraction (GEF) induced an increase in the plasma serotonin concentration in the mice. Oral administration of the GEF in mice with alcohol withdrawal decreased the immobility time in two depression-like behavioral tests and restored the alcohol withdrawal-induced serotonin decrease in plasma levels. We cannot exclude the possibility that the gintonin-mediated regulation of adrenal catecholamine release in the peripheral system, and acetylcholine and glutamate release in the central nervous system, could also contribute to the alleviation of depressive-like behaviors. The GEF-mediated attenuation of depressive-like behavior induced by alcohol withdrawal may be mediated by serotonin release from intestinal enterochromaffin cells. Therefore, the GEF might be responsible for the ginseng extract-induced alleviation of depression-related symptoms.


Immunohistochemical study of a membrane skeletal molecule, protein 4.1G, in mouse seminiferous tubules.
Protein 4.1 families have recently been established as potential organizers of an adherens system. In the adult mouse testis, protein 4.1G (4.1G) localized as a line pattern in both basal and adluminal compartments of the seminiferous tubules, attaching regions of germ cells and Sertoli cells. By double staining for 4.1G and F-actin, their localizations were shown to be different, indicating that 4.1G was localized in a region other than the basal and apical ectoplasmic specializations, which formed the Sertoli-Sertoli cell junction and Sertoli-spermatid junction, respectively. By electron microscopy, immunoreactive products were seen exclusively on the cell membranes of Sertoli cells, attaching to the various differentiating germ cells. The immunolocalization of cadherin was identical to that of 4.1G, supporting the idea that 4.1G may be functionally interconnected with adhesion molecules. In an experimental mouse model of cadmium treatment, in which tight and adherens junctions of seminiferous tubules were disrupted, the 4.1G immunostaining in the seminiferous tubules was dramatically decreased. These results indicate that 4.1G may have a basic adhesive function between Sertoli cells and germ cells from the side of Sertoli cells.


Chronic migraine.
The second edition of the International Classification of Headache Disorders revised in 2006 (ICHD-2R) gives a definition which requires 15 or more headache days per month over the past 3months with at least eight headache days per month that meet criteria for migraine without aura or that responds to migraine specific treatment. Approximately 2% of the global population suffers of chronic migraine (CM). Frequency of headache and degree of disability distinguish CM from episodic migraine (EM). There is a high frequency of medication overuse. The treatment depends on evaluation with education, lifestyle modifications, and trigger management, behavioral and pharmacologic therapies.


Analysis of collective invasion of carcinoma cells in a 3D organotypic model.
Cancer cell invasion and dissemination from primary tumors are complex multistep mechanisms which remain poorly understood. It is now clear that cancer cells can adapt their mode of invasion to the signalling provided by the surrounding stroma. Single and collective cancer cell invasion are the two invasion features most currently observed and described by pathologists. Here we describe a three-dimensional organotypic assay that allows the study of squamous cell carcinoma cell collective invasion induced by the carcinoma associated fibroblasts. This model preserves the relationship between epithelial and mesenchymal cells, which are observed in vivo, and allows to decipher the molecular and cellular mechanisms involving the tumor and its stromal microenvironment. This three-dimensional model of invasion provides an invaluable tool to gain major insights in the understanding of tumor cell dissemination.


Rheumatology experience with FDG PET / CT in inflammation of unknown origin: a single - centre report for determining factors associated with diagnostic precision.
BACKGROUND / AIM: The use of PET / CT is becoming more common in the elucidation of inflammatory processes in which the underlying cause cannot be determined by conventional examinations. Although PET / CT is an effective method for detecting inflammatory foci, the precise diagnosis may not be obtained in all cases. In addition, considering factors such as radiation exposure and cost, it becomes important to identify patients who can get results with PET / CT. In this study, it was aimed to examine the factors that can predict the differential diagnostic value of PET / CT by retrospectively scanning patients who underwent PET / CT for inflammation of unknown origin (IUO) in rheumatology practice. Demographic, clinical and laboratory information of the patients followed up in our clinic and who underwent PET / CT for differential diagnosis were enrolled. Whether they were diagnosed after PET / CT and during the follow - up period, and their diagnoses were examined. A total of 132 patients were included in the study. A previous diagnosis of rheumatic disease was present in 28.8 % of the patients, and a history of malignancy was present in 2.3 % . The patients were divided into three groups: group 1 patients with increased FDG uptake in PET / CT and diagnosis confirmed by PET / CT, group 2 patients with increased FDG uptake in PET / CT but diagnosis was not confirmed, and group 3 patients without increased FDG uptake in PET / CT. Increased FDG uptake in PET / CT was detected in 73 % of the patients. While PET / CT helped the diagnosis in 47 (35.6 %) patients (group 1), it did not help the diagnosis in 85 (64.4 %) (groups 2 and 3). Thirty - one (65.9 %) of the diagnosed patients were diagnosed with a rheumatologic disease. When the 3 groups were compared, male gender, advanced age, CRP levels, presence of constitutional symptoms, SUVmax values and number of different organs with increased FDG uptake were higher in Group 1. Sixty - six percent and 74 % of the patients in groups 2 and 3 were not diagnosed during the follow - up period. No patient in group 3 was diagnosed with malignancy during follow - up. PET / CT has high diagnostic value when combined with clinical and laboratory data in the diagnosis of IUO. Our study revealed that various factors can affect the diagnostic value of PET / CT. Similar to the literature, the statistically significant difference in CRP levels shows that patients with high CRP levels are more likely to be diagnosed with an aetiology in PET / CT. Although detection of involvement in PET / CT is not always diagnostic, there was an important finding that no malignancy was detected in the follow - up in any patient without PET / CT involvement. Key points • PET / CT is an effective method for detecting inflammatory foci. • PET / CT has proven to be effective in the diagnosis of rheumatological diseases, the extent of disease and the evaluation of response to treatment. • Indications for the use of PET / CT in the field of rheumatology and the associated factors and clinical features supporting the diagnosis with PET / CT are still to be fully clarified. • In routine practice, with PET / CT, both delays in diagnosis and examinations performed during diagnosis and the cost can be reduced.


Body fluid abnormalities in severe hyperglycemia in patients on chronic dialysis: theoretical analysis.
Hyperglycemic syndromes cause disturbances in the tonicity of body fluids, the distribution of body water between major body fluid compartments, and the external balance of body solute and water. The unique feature of dialysis-associated hyperglycemia (DH) is that, during its development, it can cause changes exclusively in the internal balance of body solute (hypertonicity) and fluids (intracellular volume contraction and extracellular volume expansion) without affecting the external balance of water or solute. This makes DH the proper substrate for studying predictions of the changes in tonicity and extracellular volume caused by hyperglycemia because these predictions fail, by and large, to account for changes in the external balance of sodium, potassium, and water observed in hyperglycemic syndromes occurring in patients with preserved renal function. The predictions suggest that the baseline state of extracellular volume and the degree of hyperglycemia are major factors influencing the magnitude of abnormalities in the tonicity and extracellular volume resulting from DH, while transfers of solute between the intracellular compartment and the extracellular compartment have relatively smaller effects. Edematous patients are at risk for greater hypertonicity and larger increases in their extracellular volume than euvolemic -- or, even less, hypovolemic -- patients with the same degree of hyperglycemia. Studies reporting the treatment of DH with only insulin therapy can be used to test these theoretical predictions and to analyze the relationship between solute and fluid abnormalities and clinical manifestations.


Inositol 1-phosphate formation in long-term potentiation and kindling.
Long-term potentiation (LTP) in the CA3 hippocampal subfield, elicited in vivo, produced significant increases in basal and in carbachol- and noradrenaline-induced hydrolysis of phosphatidylinositol-4,5-biphosphate (PtdIns(4,5)P2) as measured by the accumulation of InsP1 in hippocampal slices in vitro. Kindling, however, resulted in significant decreases in basal and in carbachol- and noradrenaline-induced accumulation of InsP1 in hippocampal slices. N-Methyl-D-aspartate (NMDA) receptors do not directly alter the hydrolysis of PtdIns(4,5)P2 in either LTP or kindling. These results demonstrate that LTP and kindling are associated with opposite alterations in efficacy of the main receptors mediating the hydrolysis of PtdIns(4,5)P2.


Development and use of active clinical decision support for preemptive pharmacogenomics.
Active clinical decision support (CDS) delivered through an electronic health record (EHR) facilitates gene-based drug prescribing and other applications of genomics to patient care. We describe the development, implementation, and evaluation of active CDS for multiple pharmacogenetic test results reported preemptively. Clinical pharmacogenetic test results accompanied by clinical interpretations are placed into the patient's EHR, typically before a relevant drug is prescribed. Problem list entries created for high-risk phenotypes provide an unambiguous trigger for delivery of post-test alerts to clinicians when high-risk drugs are prescribed. In addition, pre-test alerts are issued if a very-high risk medication is prescribed (eg, a thiopurine), prior to the appropriate pharmacogenetic test result being entered into the EHR. Our CDS can be readily modified to incorporate new genes or high-risk drugs as they emerge. Through November 2012, 35 customized pharmacogenetic rules have been implemented, including rules for TPMT with azathioprine, thioguanine, and mercaptopurine, and for CYP2D6 with codeine, tramadol, amitriptyline, fluoxetine, and paroxetine. Between May 2011 and November 2012, the pre-test alerts were electronically issued 1106 times (76 for thiopurines and 1030 for drugs metabolized by CYP2D6), and the post-test alerts were issued 1552 times (1521 for TPMT and 31 for CYP2D6). Analysis of alert outcomes revealed that the interruptive CDS appropriately guided prescribing in 95% of patients for whom they were issued. Our experience illustrates the feasibility of developing computational systems that provide clinicians with actionable alerts for gene-based drug prescribing at the point of care.


Hyperthermic intraperitoneal chemotherapy (HIPEC) and cytoreductive surgery (CRS) in ovarian cancer: A systematic review and meta-analysis.
Emerging evidence suggests that hyperthermic intraperitoneal chemotherapy (HIPEC) with cytoreductive surgery (CRS) shows a survival benefit over CRS alone for patients with epithelial ovarian carcinoma (EOC). This systematic review and meta-analysis will assess the safety and efficacy of HIPEC with CRS for EOC. Searches of five databases from inception to 17/02/15 was performed. Clinical outcomes were synthesised, with full tabulation of results. A total of 9 comparative studies and 28 studies examining HIPEC + CRS for primary and/or recurrent EOC were included. Meta-analysis of the comparative studies showed HIPEC + CRS + chemotherapy had significantly better 1-year survival compared with CRS + chemotherapy alone (OR: 3.76, 95% CI 1.81-7.82). The benefit of HIPEC + CRS continued for 2-, 3-, 4-, 5- and 8-year survival compared to CRS alone (OR: 2.76, 95% CI 1.71-4.26; OR: 5.04, 95% CI 3.24-7.85; OR: 3.51, 95% CI 2.00-6.17; OR: 3.46 95% CI 2.19-5.48; OR: 2.42, 95% 1.38-4.24, respectively). Morbidity and mortality rates were similar. Pooled analysis of all studies showed that among patients with primary EOC, the median, 1-, 3-, and 5-year overall survival rates are 46.1 months, 88.2%, 62.7% and 51%. For recurrent EOC, the median, 1-, 3-, and 5-year overall survival rates are 34.9 months, 88.6%, 64.8% and 46.3%. A step-wise positive correlation between completeness of cytoreduction and survival was found. The addition of HIPEC to CRS and chemotherapy improves overall survival rates for both primary and recurrent EOC.


Effectiveness of the CAGE questionnaire, gamma-glutamyltransferase and mean corpuscular volume of red blood cells as markers for alcohol-related problems in the workplace.
To evaluate the usefulness of gamma-glutamyltransferase (GGT) and mean corpuscular volume (MCV), as well as that of the CAGE questionnaire, in workplace screening for alcohol abuse/dependence. A total of 183 male employees were submitted to structured interviews (Structured Clinical Interview for DSM-IV 2.0 and CAGE questionnaire). Blood samples were collected. Diagnostic accuracy and odds ratio were determined for the CAGE, GGT and MCV. The CAGE questionnaire presented the best sensitivity for alcohol dependence (91%; specificity, 87.8%) and for alcohol abuse (87.5%, specificity, 80.9%), which increased when the questionnaire was used in combination with GGT (sensitivity, 100% and 87.5%, respectively; specificity, 68% and 61.5, respectively). CAGE positive results and/or alterations in GGT were less likely to occur among employees not presenting alcohol abuse/dependence than among those presenting such abuse (OR for CAGE=13, p<0.05; OR for CAGE-GGT=11, p<0.05) or dependence (OR for CAGE=76, p<0.01; OR for GGT=5, p<0.01). Employees not presenting alcohol abuse/dependence were also several times more likely to present negative CAGE or GGT results. The use short, simple questionnaires, combined with that of low-cost biochemical markers, such as GGT, can serve as an initial screening for alcohol-related problems, especially for employees in hazardous occupations. The data provided can serve to corroborate clinical findings.


Radiotherapy-induced salivary dysfunction: Structural changes, pathogenetic mechanisms and therapies.
This review addressed the structural changes observed in salivary glands and pathogenic mechanisms resulting from oxidative stress caused by radiotherapy. The preventive and regenerative therapies for altered acinar morphology and glandular function were also reviewed. Among acute and late microscopic alterations in glandular tissue, there are particularly changes indicative of cell death, hypovascularization, formation of fibrous tissue and edema. A critical role was identified for the Akt-MDM2-p53 pathway in the suppression of DNA damage-induced apoptosis in acinar cells. Prophylactic treatment with pilocarpine, cevilemine, bethanechol and isoproterenol has shown a positive effect on salivary flow, but lasting results have not been observed. Growth factors, histamine and lidocaine, have also demonstrated radioprotective effects on the salivary glands. Stem cell preservation and transplantation may be an alternative to maintain tissue homeostasis and thus allow glandular regeneration. Knowledge of the structural changes observed in the salivary glands contributes to proving the short- and long-term efficacy of the therapies investigated. It is important to know the mechanisms involved in radiation-induced damage, since the control of the pathogenic mechanisms can inhibit the initial process of tissue degeneration. The challenge for investigators is to protect normal cells selectively, without promoting tumor growth.


Possible target neurons of the reticulospinal cholecystokinin (CCK) projection to the lamprey spinal cord: immunohistochemistry combined with intracellular staining with lucifer yellow.
A subpopulation of reticulospinal neurons in the posterior rhombencephalic reticular nucleus in lamprey contains a cholecystokinin (CCK)-like peptide. Varicosities of these neurons, visualized by immunohistofluorescence, were found to be in close apposition to Lucifer yellow-filled spinal motoneurons and giant relay interneurons. Certain other types of interneurons and primary sensory neurons had no, or very few, close contacts with CCK-immunoreactive boutons.


APJ expression is lost in isolated embryonic coronary endothelial cell culture in vitro.
Coronary artery disease is one of the leading causes of death worldwide, and yet we lack the appropriate therapeutic treatments for it. Investigation into the mechanisms of coronary vessel development can provide insights into potential therapies to repair and regenerate damaged coronary arteries. Our previous study in the mouse embryo have implicated APJ, a G-protein coupled receptor that is expressed by coronary endothelial cells in vivo, to be an important regulator of coronary vessel development. In this study, we report an unexpected finding that the isolated embryonic coronary endothelial cells lose APJ expression in culture in vitro.


Chemical chaperones assist intracellular folding to buffer mutational variations.
Hidden genetic variations have the potential to lead to the evolution of new traits. Molecular chaperones, which assist protein folding, may conceal genetic variations in protein-coding regions. Here we investigate whether the chemical milieu of cells has the potential to alleviate intracellular protein folding, a possibility that could implicate osmolytes in concealing genetic variations. We found that the model osmolyte trimethylamine N-oxide (TMAO) can buffer mutations that impose kinetic traps in the folding pathways of two model proteins. Using this information, we rationally designed TMAO-dependent mutants in vivo, starting from a TMAO-independent protein. We show that different osmolytes buffer a unique spectrum of mutations. Consequently, the chemical milieu of cells may alter the folding pathways of unique mutant variants in polymorphic populations and lead to unanticipated spectra of genetic buffering.


Causal effects of serum sex hormone binding protein levels on the risk of amyotrophic lateral sclerosis: a mendelian randomization study.
Extensive observational studies have suggested an association between serum sex-hormone binding globulin (SHBG) and Alzheimer's disease (AD); however, causality remains unclear. Furthermore, the effects on other neurodegenerative diseases have been poorly investigated. We aimed to explore the causal effects of genetically predicted SHBG serum levels on common neurodegenerative diseases. A two-sample Mendelian randomization (MR) approach was used. Genetic variants of SHBG levels in the serum, detected using the chemiluminescent two-step sandwich immunoassay method, were identified from a genome-wide association meta-analysis from the UK Biobank (N=363,228). Summary-level data for AD, and other common neurodegenerative diseases including Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), dementia with Lewy bodies (DLB), and frontotemporal dementia (FTD) were adopted from the corresponding large genome-wide association studies of individuals of European ancestry, which were either clinically or autopsy-diagnosed. Causal estimates were calculated using the inverse-variance weighted (IVW) method and several sensitivity methods (MR-Egger, weighted median, and weighted mode). Egger intercept, MR-PRESSO, and leave-one-out analyses were used to identify potential violations. Genetically determined serum SHBG levels [odds ratio (ORIVW) =1.113, 95% CI: 1.019-1.215, P=0.017] were associated with an increased risk of ALS. This causal effect was confirmed using sensitivity analyses, including the MR-Egger (OR =1.229, 95% CI: 1.049-1.441, P=0.012), weighted median (OR =1.231, 95% CI: 1.077-1.406, P=0.002), and weighted mode (OR =1.235, 95% CI: 1.067-1.431, P=0.005) methods. No notable heterogeneity or directional pleiotropy was observed. However, leave-one-out analysis showed that rs9892297 drove the observed effects. There was no evidence that genetically predicted serum SHBG levels affect other neurodegenerative diseases, including AD, PD, MS, DLB, and FTD (all P>0.05). This MR analysis found that genetically determined serum SHBG was associated with an increased risk of ALS rather than AD, which is inconsistent with previous observational studies. This novel finding highlights the potential of SHBG in peripheral serum for ALS prevention. Further research into the effects of SHBG on other neurodegenerative diseases is required, especially because of the increased utilization of hormone therapy.


Metal versus plastic stents for malignant biliary obstruction: an update meta-analysis.
The data on comparisons of stent patency, re-intervention rate and patient survival between metal and plastic stents in palliation of malignant biliary obstruction have never been pooled. We carry out a meta-analysis to summarise current evidence for clinical efficacy of metal and plastic stents in the treatment of malignant biliary obstruction. A comprehensive search of several databases was conducted. A fixed-effects or random-effects model was used to pool data of all study endpoints. Sensitivity analysis and subgroup analysis (distal vs. hilar biliary obstruction) were also performed. Ten randomized clinical trials were identified. Compared with plastic stents, metal stents were associated with a significantly longer stent patency (HR=0.36; 95% CI: 0.28-0.47; I(2)=0%), fewer numbers of re-intervention (WMD=0.59; 95% CI: 0.28-0.90; I(2)=76.4%) and longer patient survival (HR=0.74; 95% CI: 0.64-0.85; I(2)=16.0%). These results were still significant by sensitivity analysis. All outcomes reached statistical significance except of the pooled WMD of number of re-intervention in the studies with hilar biliary obstruction. No publication bias was observed. Metal stents were associated with a significantly longer stent patency, lower re-intervention rate and longer patient survival in palliation of malignant biliary obstruction when compared to plastic stents.


Rapid and sensitive method for the analysis of carbon monoxide in blood using gas chromatography with flame ionisation detection.
In order to measure changes in physiological CO concentrations in blood with good accuracy, a method was developed using gas chromatography with flame ionisation detection (250 degrees C). A nickel catalyst system was fitted to convert CO to methane at 375 degrees C after separation with a molecular sieve column at 35 degrees C. Helium was used as carrier at 30 ml/min. Porcine or human blood (400 microl) was sampled in gastight tubes and treated with sulfuric acid and saponin (800 microl). Accuracy was 1.4% and 1.5% (RSD), respectively. Precision was 2.8% (porcine blood). Limit of detection was 0.01 nmol/ml gas and limit of quantification 12 nmol/ml blood. Calibration was made in the interval 12-514 nmol/ml blood (corresponding to 0.1-6% COHb). Samples were stable for at least a month at +4 degrees C. This paper describes a method with high sensitivity and good accuracy, suitable for analysis of low CO concentrations.


Critical Calibration of Mass Photometry for Higher-Mass Samples Such as Adeno-Associated Virus Vectors.
Mass photometry (MP) is a label-free, single-molecule technique that can determine molecular mass distribution with very low sample consumption in a short time. Because of the established experimental instrument and analytical software, MP measurements may be readily obtained; thus, the application of MP is expanding, especially in the fields of bioscience and biotechnology. However, because the MP data quality is strongly focus-dependent, optical settings must be intrinsically strict. In this study, we report the importance of the critical calibration of the mass photometer, which is required for the accurate estimation of high-molecular mass samples, such as adeno-associated virus vectors. Additionally, a method for optimizing the instrument settings, including the calibration of the stage, is presented.


Characteristics and prognosis of resected T3 non-small cell lung cancer.
T3 tumors can be divided into several subgroups depending on the type of anatomical structure invaded: chest wall, mediastinal pleura, or main bronchus. The aim of this study was to analyze the characteristics and prognosis of each subgroup of T3 tumors. The results of surgical treatment were retrospectively analyzed for 261 patients with T3 non-small cell lung cancer invading either the mediastinal pleura or parietal pericardium by direct extension (mediastinal pT3, n = 68), or main bronchus (bronchial pT3, n = 68), or chest wall (chest wall pT3, n = 125) that were operated on between 1984 and 1996. Complete resection including radical mediastinal lymph node dissection was intended in all patients. One patient had segmentectomy, 91 had lobectomy (34.9%), and 169 had pneumonectomy (64.8%). One hundred and fifty-eight patients received adjuvant radiation therapy and 7 patients received both adjuvant chemotherapy and radiation therapy. Actuarial survival curves were drawn using the Kaplan-Meier method and risk factors for late death were identified. In-hospital mortality was 6.1%. Follow-up was 98% complete. Global 5-year survival was 28%, with survival being not significantly different among the three subgroups: 34.9%, 30.6%, and 22.5% (p = 0.19) in the bronchial pT3, mediastinal pT3, and chest wall pT3 subgroups, respectively. Resection margins were microscopically invaded in 33 patients (12.6%). Seventy-four patients had N1 involvement (28.4%) and 78 patients had N2 involvement (29.8%). N0 involvement was more prevalent in the chest wall pT3 subgroup, whereas N1 involvement was more prevalent in the bronchial pT3 subgroup and N2 involvement was more prevalent among patients with mediastinal invasion. Pathologic factors influencing the 5-year survival were tumor size (p = 0.03) and N involvement (p = 0.003). Histology, type of surgical resection (lobectomy versus pneumonectomy), and use of adjuvant therapy did not influence survival significantly. Five-year survival was not significantly different among the three subgroups of pT3 non-small cell lung cancer, although bronchial pT3 tumors tended to have a better prognosis and chest wall pT3 tumors tended to have a worse prognosis. The pathologic characteristics of each pT3 subgroup seems different. Further research is warranted to explore the pathologic and biological factors influencing prognosis for each pT3 subgroup.


Lissencephaly in Shih Tzu dogs.
Lissencephaly is a brain malformation characterized by smooth and thickened cerebral surface, which may result in structural epilepsy. Lissencephaly is not common in veterinary medicine. Here, we characterize the first cases of lissencephaly in four Shih Tzu dogs, including clinical presentations and findings of magnetic resonance imaging of lissencephaly and several concomitant brain malformations. Early-onset acute signs of forebrain abnormalities were observed in all dogs, which were mainly cluster seizures and behavioral alterations. Based on neurological examination, the findings were consistent with symmetrical and bilateral forebrain lesions. Metabolic disorders and inflammatory diseases were excluded. Magnetic resonance imaging for three dogs showed diffuse neocortical agyria and thickened gray matter while one dog had mixed agyria and pachygyria. Other features, such as internal hydrocephalus, supracollicular fluid accumulation, and corpus callosum hypoplasia, were detected concomitantly. Antiepileptic drugs effectively controlled cluster seizures, however, sporadic isolated seizures and signs of forebrain abnormalities, such as behavioral alterations, central blindness, and strabismus persisted. Lissencephaly should be considered an important differential diagnosis in Shih Tzu dogs presenting with early-onset signs of forebrain abnormalities, including cluster seizures and behavioral alterations. Magnetic resonance imaging was appropriate for ante-mortem diagnosis of lissencephaly and associated cerebral anomalies.


Is the impaired flow after hypospadias correction due to increased urethral stiffness?
A low flow rate without clinical symptoms is commonly found in boys after hypospadias correction. Urethral calibration usually shows no abnormalities. We investigated whether this impairment might be caused by increased neo-urethral wall stiffness. From polyvinyl alcohol cryogel two models of the urethra were made, hypospadias and control; both had a constant and equal inner diameter and equal compliance. The hypospadias model had a less compliant distal segment mimicking the distal neo-urethra after hypospadias correction. In both models, flow rate was recorded as a function of bladder pressure. To test whether the length of the less compliant segment had an effect on the flow rate, both models were shortened by cutting off 1-cm segments. In a physiological range of bladder pressures (10-130 cmH(2)O) the mean flow rate (+/-1 SEM) in the hypospadias model was 2.8+/-0.3 ml/s, significantly lower (P<0.05) than in the control model (5.4+/-0.6 ml/s). Shortening of the hypospadias model showed some increase in flow rate, but this was not statistically significant. In the control model there was also no significant variation in flow rate. A low-compliant segment of a urethral model reduced the flow rate. Extrapolating these results to asymptomatic boys with a low urinary flow rate after hypospadias repair might justify a watchful waiting policy.


Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection marker.
Beauveria bassiana has been investigated for use in the biological control of several insects in agricultural practice. To understand the molecular basis of virulence and host specificity and to improve the entomopathogenicity of B. bassiana, we have developed a simple, highly efficient and reliable Agrobacterium-mediated transformation method for B. bassiana using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. Most transformants contained single copies of T-DNA and the T-DNA inserts were stably inherited after five generations. With this highly efficient transformation method for B. bassiana, we also obtained two putative T-DNA-tagged mutants that may have altered growth habits or virulence. Thus, the described protocol could provide a useful tool to manipulate the genetic make-up and to tag genes that may be important for virulence or growth and development of B. bassiana.


Comparison of antigenicity among haemocytes of seven bivalve species by monoclonal antibodies against haemocytes of scallop (Chlamys farreri).
In this paper, haemocyte antigenicity of seven bivalve species (scallop (Chlamys farreri), bay scallop (Argoecten irradians), oyster (Crassostrea talienwhanensis), asiatic hard clam (Meretrix meretrix), monila clam (Ruditapes philipinarum), purplish washington clam (Saxidomus purpuratus) and horny ark (Scapharca subcrenta)) were analysed using monoclonal antibodies (MAbs) 1E7, 1F12, 2C6 and 2H5 against haemocytes of C. farreri, employed methods of immuno-dotblotting (IDB), indirect immunofluorescence assay (IIFA) and western-blotting (WB). The four MAbs react with haemocytes of seven bivalve species. As the results for both IDB and IIFA, MAb 1E7 was positive with haemocytes of R. philipinarum, MAb 1F12 with haemocytes of A. irradians, M. meretrix, R. philipinarum and S. purpuratus; MAb 2C6 with haemocytes of the other five bivalve species except for S. purpuratus. MAb 2H5 was negative with haemocytes of the other six bivalve species in IDB, but was positive with haemocytes of R. philipinarum and S. purpuratus in IIFA. Further experiments by WB showed MAb 1F12 was able to recognise the protein of A. irradians haemocyte at molecular weights of 156 and 80 kDa, haemocytes of M. meretris, R. philipinarum, S. purpuratus, at 60, 30, 58 kDa, respectively. MAb 2C6 recognised haemocyte M. meretris proteins at 50 and 37 kDa, A. irradians, C. talienwhanensis, R. philipinarum, S. subcrenta at 40, 38, 38, 45 kDa, respectively. There were no protein bands reacting with MAb 1E7 and MAb 2H5. The results indicate antigenic similarities exist among haemocytes of the seven bivalve species.


Accuracy of medical models made by additive manufacturing (rapid manufacturing).
Additive manufacturing (AM) is being increasingly used for producing medical models. The accuracy of these models varies between different materials, AM technologies and machine runs. To determine the accuracy of selective laser sintering (SLS), three-dimensional printing (3DP) and PolyJet technologies in the production of medical models. 3D skull models: "original", "moderate" and "worse". SLS, 3DP and PolyJet models, and a coordinate measuring machine (CMM). Measuring balls designed for measurements were attached to each 3D model. Skull models were manufactured using SLS, 3DP and PolyJet. The midpoints of the balls were determined using CMM. The distances between these points were calculated and compared with the 3D model. The dimensional error for the PolyJet was 0.18 ± 0.12% (first measurement) and 0.18 ± 0.13% (second measurement), for SLS 0.79 ± 0.26% (first model) and 0.80 ± 0.32% (second model), and for 3DP 0.67 ± 0.43% (original model, first measurement) and 0.69 ± 0.44% (original model, second measurement), 0.38 ± 0.22% (moderate model) and 0.55 ± 0.37% (worse model). Repeatability of the measurement method was 0.12% for the PolyJet and 0.08% for the 3DP. A novel measuring technique was developed and its repeatability was found to be good. The accuracy of the PolyJet was higher when compared with SLS or 3DP.


[Enzyme-histochemical examinations of prostatic adenofibromatosis. I. Enzyme activities of glucose metabolism].
In the present paper is reported on the histochemical demonstration of reference enzymes of the glucose metabolism in adenofibromyomatosis prostatae. Here non-glycolitic metabolic processes were particularly taken into consideration. The results do not show any restriction of the glucose metabolism in the benign growth process.


Diffuse lung uptake of technetium-99m sulfur colloid associated with fatty liver disease of pregnancy.
Diffuse pulmonary uptake of sulfur colloid was seen in a 35-year-old postpartum white female with the presumptive diagnosis of fatty liver disease of pregnancy. As the disease process resolved, the pulmonary uptake of sulfur colloid disappeared. To the authors' knowledge, this scan association has not been previously reported.


Chronic Psychological Stress Accelerates Vascular Senescence and Impairs Ischemia-Induced Neovascularization: The Role of Dipeptidyl Peptidase-4/Glucagon-Like Peptide-1-Adiponectin Axis.
Exposure to psychosocial stress is a risk factor for cardiovascular disease, including vascular aging and regeneration. Given that dipeptidyl peptidase-4 (DPP4) regulates several intracellular signaling pathways associated with the glucagon-like peptide-1 (GLP-1) metabolism, we investigated the role of DPP4/GLP-1 axis in vascular senescence and ischemia-induced neovascularization in mice under chronic stress, with a special focus on adiponectin -mediated peroxisome proliferator activated receptor-γ/its co-activator 1α (PGC-1α) activation. Seven-week-old mice subjected to restraint stress for 4 weeks underwent ischemic surgery and were kept under immobilization stress conditions. Mice that underwent ischemic surgery alone served as controls. We demonstrated that stress impaired the recovery of the ischemic/normal blood-flow ratio throughout the follow-up period and capillary formation. On postoperative day 4, stressed mice showed the following: increased levels of plasma and ischemic muscle DPP4 and decreased levels of GLP-1 and adiponectin in plasma and phospho-AMP-activated protein kinase α (p-AMPKα), vascular endothelial growth factor, peroxisome proliferator activated receptor-γ, PGC-1α, and Sirt1 proteins and insulin receptor 1 and glucose transporter 4 genes in the ischemic tissues, vessels, and/or adipose tissues and numbers of circulating endothelial CD31+/c-Kit+ progenitor cells. Chronic stress accelerated aortic senescence and impaired aortic endothelial sprouting. DPP4 inhibition and GLP-1 receptor activation improved these changes; these benefits were abrogated by adiponectin blocking and genetic depletion. These results indicate that the DPP4/GLP-1-adiponectin axis is a novel therapeutic target for the treatment of vascular aging and cardiovascular disease under chronic stress conditions.


Editorial on Special Issue "Advances and Novel Treatment Options in Metastatic Melanoma".
Investigating mechanisms controlling melanoma setup, development and progression is currently an extremely hot and rapidly evolving topic [...].