Comprehensive metabolic profiling of Parkinson's disease by liquid chromatography-mass spectrometry.
Parkinson's disease (PD) is a prevalent neurological disease in the elderly with increasing morbidity and mortality. Despite enormous efforts, rapid and accurate diagnosis of PD is still compromised. Metabolomics defines the final readout of genome-environment interactions through the analysis of the entire metabolic profile in biological matrices. Recently, unbiased metabolic profiling of human sample has been initiated to identify novel PD metabolic biomarkers and dysfunctional metabolic pathways, however, it remains a challenge to define reliable biomarker(s) for clinical use. We presented a comprehensive metabolic evaluation for identifying crucial metabolic disturbances in PD using liquid chromatography-high resolution mass spectrometry-based metabolomics approach. Plasma samples from 3 independent cohorts (n = 460, 223 PD, 169 healthy controls (HCs) and 68 PD-unrelated neurological disease controls) were collected for the characterization of metabolic changes resulted from PD, antiparkinsonian treatment and potential interferences of other diseases. Unbiased multivariate and univariate analyses were performed to determine the most promising metabolic signatures from all metabolomic datasets. Multiple linear regressions were applied to investigate the associations of metabolites with age, duration time and stage of PD. The combinational biomarker model established by binary logistic regression analysis was validated by 3 cohorts. A list of metabolites including amino acids, acylcarnitines, organic acids, steroids, amides, and lipids from human plasma of 3 cohorts were identified. Compared with HC, we observed significant reductions of fatty acids (FFAs) and caffeine metabolites, elevations of bile acids and microbiota-derived deleterious metabolites, and alterations in steroid hormones in drug-naïve PD. Additionally, we found that L-dopa treatment could affect plasma metabolome involved in phenylalanine and tyrosine metabolism and alleviate the elevations of bile acids in PD. Finally, a metabolite panel of 4 biomarker candidates, including FFA 10:0, FFA 12:0, indolelactic acid and phenylacetyl-glutamine was identified based on comprehensive discovery and validation workflow. This panel showed favorable discriminating power for PD. This study may help improve our understanding of PD etiopathogenesis and facilitate target screening for therapeutic intervention. The metabolite panel identified in this study may provide novel approach for the clinical diagnosis of PD in the future.


Purinergic signalling underlies transforming growth factor-β-mediated bladder afferent nerve hyperexcitability.
The sensory components of the urinary bladder are responsible for the transduction of bladder filling and are often impaired with neurological injury or disease. Elevated extracellular ATP contributes, in part, to bladder afferent nerve hyperexcitability during urinary bladder inflammation or irritation. Transforming growth factor-β1 (TGF-β1) may stimulate ATP release from the urothelium through vesicular exocytosis mechanisms with minimal contribution from pannexin-1 channels to increase bladder afferent nerve discharge. Bladder afferent nerve hyperexcitability and urothelial ATP release with CYP-induced cystitis is decreased with TGF-β inhibition. These results establish a causal link between an inflammatory mediator, TGF-β, and intrinsic signalling mechanisms of the urothelium that may contribute to the altered sensory processing of bladder filling. The afferent limb of the micturition reflex is often compromised following bladder injury, disease and inflammatory conditions. We have previously demonstrated that transforming growth factor-β (TGF-β) signalling contributes to increased voiding frequency and decreased bladder capacity with cystitis. Despite the functional presence of TGF-β in bladder inflammation, the precise mechanisms of TGF-β mediating bladder dysfunction are not yet known. Thus, the present studies investigated the sensory components of the urinary bladder that may underlie the pathophysiology of aberrant TGF-β activation. We utilized bladder-pelvic nerve preparations to characterize bladder afferent nerve discharge and the mechanisms of urothelial ATP release with distention. Our findings indicate that bladder afferent nerve discharge is sensitive to elevated extracellular ATP during pathological conditions of urinary bladder inflammation or irritation. We determined that TGF-β1 may increase bladder afferent nerve excitability by stimulating ATP release from the urothelium via vesicular exocytosis mechanisms with minimal contribution from pannexin-1 channels. Furthermore, blocking aberrant TGF-β signalling in cyclophosphamide-induced cystitis with TβR-1 inhibition decreased afferent nerve hyperexcitability with a concomitant decrease in urothelial ATP release. Taken together, these results establish a role for purinergic signalling mechanisms in TGF-β-mediated bladder afferent nerve activation that may ultimately facilitate increased voiding frequency. The synergy between intrinsic urinary bladder signalling mechanisms and an inflammatory mediator provides novel insight into bladder dysfunction and supports new avenues for therapeutic intervention.


Ifosfamide bolus followed by five days continuous infusion in extensively pretreated patients with advanced breast cancer: a phase II study.
A phase II study with ifosfamide in pretreated patients with advanced breast cancer was performed to determine the objective response rate, the toxicity and the feasibility of the regimen. Patients enrolled had advanced breast cancer pretreated with at least one previous regimen of chemotherapy for advanced disease. Treatment consisted of ifosfamide infused at a dose of 2 g/m2 iv in 4 hrs followed by ifosfamide, 8 g/m2 iv in 120 hrs in ambulatory treatment, using a portable external pump system. The total dose of ifosfamide was 10 g/m2; mesna (4 g/m2 iv) was administered mixed with ifosfamide in 120 hrs Cycles were repeated every 3 weeks. Three patients were pretreated with neoadjuvant and 15 with adjuvant chemotherapy. All patients were treated for advanced disease (median number of regimens, 1; range, 1-3): 21 with the cyclophosphamide-containing regimen and 15 with adryamicin. Sixteen patients received one or more lines of endocrine therapy. Fifteen patients had dominant site in viscera, 6 in bone, and only one in soft tissue; 17 patients had more than one site of disease. Twenty-two patients were enrolled and all were assessable for response and toxicity. A partial response was reached in 5 patients (23%; 95% confidence limits 5% to 60%). Hematologic toxicity was the dose-limiting side effect; grade 4 leukopenia occurred in 10 patients (46%). Considering the response rate obtained in our series of intensively pretreated patients, the results seem to indicate that the regimen is active and could be included among the possible options in the treatment of patients with refractory, poor-prognosis, advanced breast carcinoma.


Impact of disease duration on proteomic bioprofile and prognosis in rheumatoid arthritis patients.
Cardiovascular disease worsens the prognosis of rheumatoid arthritis (RA) and vice-versa. Inflammation may be a common pathway for both conditions. It is expected that a longer RA duration leads to a greater inflammatory cumulative exposure burden; however, studies on the association between RA disease duration and outcomes are scarce. Our aim is to compare the characteristics, biomarker expression and outcomes according to the duration of RA. Prospective cohort study including 399 RA patients, with detailed clinical, echocardiographic, and proteomic phenotyping that were compared across tertiles of RA disease duration. Cox proportional models were used to study the association of disease duration with cardiovascular outcomes. RA duration tertiles were: tertile 1 with median of 3.2; tertile 2 with median of 8.8; and tertile 3 with median of 21.8 years. Compared to tertile 1, patients in tertile 3 were older, had more erosive disease, more frequent echocardiographic alterations, lower haemoglobin and walked a shorter distance on the 6MWT. Natriuretic peptides, cathepsin L1, galectin 9, matrix metalloproteinase-12, adrenomedullin and tumour necrosis factor receptor 11A were higher in patients with longer disease duration. Compared to patients in tertile 1, those in tertile 3 had higher risk of a subsequent cardiovascular hospitalisation or cardiovascular death (HR 2.71, 95%CI 1.06-6.92, p=0.04). RA patients with longer disease duration had more organ damage and worse outcomes than those with shorter disease duration. Biomarker expression suggested that patients with longer RA duration had activation of pathways related to inflammation, extracellular matrix organisation, fibrosis and congestion.


Transient alkalinization of the leaf apoplast stiffens the cell wall during onset of chloride salinity in corn leaves.
During chloride salinity, the pH of the leaf apoplast (pHapo) transiently alkalizes. There is an ongoing debate about the physiological relevance of these stress-induced pHapo changes. Using proteomic analyses of expanding leaves of corn (Zea mays L.), we show that this transition in pHapo conveys functionality by (i) adjusting protein abundances and (ii) affecting the rheological properties of the cell wall. pHapo was monitored in planta via microscopy-based ratio imaging, and the leaf-proteomic response to the transient leaf apoplastic alkalinization was analyzed via ultra-high performance liquid chromatography-MS. This analysis identified 1459 proteins, of which 44 exhibited increased abundance specifically through the chloride-induced transient rise in pHapo These elevated protein abundances did not directly arise from high tissue concentrations of Cl- or Na+ but were due to changes in the pHapo Most of these proteins functioned in growth-relevant processes and in the synthesis of cell wall-building components such as arabinose. Measurements with a linear-variable differential transducer revealed that the transient alkalinization rigidified (i.e. stiffened) the cell wall during the onset of chloride salinity. A decrease in t-coumaric and t-ferulic acids indicates that the wall stiffening arises from cross-linkage to cell wall polymers. We conclude that the pH of the apoplast represents a dynamic factor that is mechanistically coupled to cellular responses to chloride stress. By hardening the wall, the increased pH abrogates wall loosening required for cell expansion and growth. We conclude that the transient alkalinization of the leaf apoplast is related to salinity-induced growth reduction.


[Lung cancer and age factor].
The authors provide the results of examination and treatment of 920 young patients under 45 (group I) and 462 elderly patients aged 60-74 years (group II). It has been revealed that in the young patients, primarily affected were the distal, proximal, intermediate and main bronchi. In group I patients, the tumor was localized in the intermediate or in the main (extrapulmonary) bronchus (in 6.9 and 9.8% of cases, respectively). In the elderly patients, the analogous data accounted for 2.7 and 1%, respectively. The segmental bronchi were more often involved in the patients of group II (11.3%) than in those of group I (5.3% of the cases examined). Analysis of the forms of lung carcinoma shows that in group I patients, the tumor was more often located near the root. Thus, in the young patients, the nodular peribronchial and peripheral (connected with the bronchus gap) forms of growth have been recorded in 34.1 and 25% of the cases examined whereas in the elderly patients, only in 14 and 13.1% of the cases. It has been noted that group I patients mostly demonstrated poorly differentiated carcinoma (47.6%) rather than high-differentiated epidermoid cancer (35.8%). Meanwhile high-differentiated adenocarcinoma was seen only in 16.4% of the cases analyzed. In the elderly patients, the estimates were respectively 29.6, 59.0 and 11.2%. It should be emphasized that in young male patients, poorly differentiated carcinoma occurred 1.7 times more often that in group II patients (48.8 and 28.3%, respectively).


Human melanoma and leukemia associated antigens difined by nonhuman primate antisera.
The rationale of our approach to the detection and characterization of human leukemia and melanoma associated membrane antigens is to take advantage of the unique immunological recognition perspective of simians to huamn antigens. There are enough antigenic similarities between man and nonhuman primates to blunt the immune response of apes and monkeys to huamn species-specific antigens. Their response to allogeneic normal human antigens such as HL-A can now be managed by in vitro absorption procedures. High titered antibodies specific for tumor associated membrane antigens have not been well documented in serological studies with patients' sera. Immunization of nonhuman primates utilizes a fully immunocompetent recipient who has no tumor and is not on chemotherapy. The immunization can utilize adjuvants and other regimens that morally and ethically cannot be easily used for human allogeneic immunization. The simian antisera that are shown to be specific for human tumor membrane antigens can then be utilized for studies on the isolation and characterization of the antigens. Similar studies with HL-A antigens indicated that the solubilized partially purified antigens can then be used to elicit high titered antibodies in other simians. These latter antisera can then be used for more sensitive assays and studies on the association of the antigen(s) with the cell membrane. Although the work summarized here covers leukemia and melamona antigens, it is felt the approach will serve as a model for the detection by simian antisera of tumor-specific membrane antigens of cancer cells from patients with various types of solid tumors.


Cognitive dysfunction and mild closed head injury in traumatic spinal cord injury.
Previous investigators have reported that 25%-50% of traumatic spinal cord injury (SCI) patients sustain loss of consciousness (LOC), posttraumatic amnesia (PTA), or both concurrently with their SCI. A LOC of 20 minutes or less, or a PTA of 24 hours has been associated with prolonged time off work, and deficits in attention, concentration, memory, and judgment. Consequently, many traumatic SCI patients may also be the victims of a concomitant closed head injury (CHI) with cognitive sequelae. To test this hypothesis, a prospective study was conducted of 30 consecutive trauma-related SCI patients admitted to the Rehabilitation Institute of Chicago to determine the incidence of CHI and cognitive dysfunction (CD). The Halstead Category Test (HCT) was administered to each patient between 8 and 12 weeks after injury, and was considered abnormal if the patient committed 51 or more errors. Fifty-seven percent (n = 11) of all patients had abnormal HCT scores suggestive of higher level cognitive dysfunction. Patients with a new CHI had a mean HCT score of 65.2, as compared to 63.5 for patients with a premorbid CHI, and 46.3 for patients without a history of CHI. Although there was a trend toward higher HCT scores in patients who had any history of CHI, these differences did not reach statistical significance. The results of this study suggest that many trauma-related SCI patients are at risk for CD eight weeks after injury. Such cognitive abnormalities would be expected to impede rehabilitation and retard the requisite learning of new skills.


Fast and practical algorithms for planted (l, d) motif search.
We consider the planted (l, d) motif search problem, which consists of finding a substring of length l that occurs in a set of input sequences {s1, . . . , sn} with up to d errors, a problem that arises from the need to find transcription factor-binding sites in genomic information. We propose a sequence of practical algorithms, which start based on the ideas considered in PMS1. These algorithms are exact, have little space requirements, and are able to tackle challenging instances with bigger d, taking less time in the instances reported solved by exact algorithms. In particular, one of the proposed algorithms, PMSprune, is able to solve the challenging instances, such as (17, 6) and (19, 7), which were not previously reported as solved in the literature.


[Ultrasound characteristics of uterine myoma before and after uterine artery embolization].
The diagnosis and treatment of uterine leiomyoma are topical problems of modern gynecology and radiodiagnosis. Organ-saving treatments for uterine myoma, one of which is uterine artery embolization, are gaining wide acceptance now. The objective of the study was to increase the informative value of ultrasound study to predict the uterine myoma after uterine artery embolization. One hundred uterine myoma patients aged 20 to 52 years were examined. Small pelvic Doppler ultrasonography was carried out in all the patients. The reduction of myomatous nodules was estimated after uterine artery embolization. The decrease in uterine myoma sizes was found to be due to the reduction in their vascularization and the occurrence of ischemia with degeneration in the myoma. Ultrasonography was found to be most accessible and informative in the prognostic and postoperative evaluation of the efficiency of X-ray endovascular treatment for uterine myoma.


Charge-dependent interactions of monomeric and filamentous actin with lipid bilayers.
The cytoskeletal protein actin polymerizes into filaments that are essential for the mechanical stability of mammalian cells. In vitro experiments showed that direct interactions between actin filaments and lipid bilayers are possible and that the net charge of the bilayer as well as the presence of divalent ions in the buffer play an important role. In vivo, colocalization of actin filaments and divalent ions are suppressed, and cells rely on linker proteins to connect the plasma membrane to the actin network. Little is known, however, about why this is the case and what microscopic interactions are important. A deeper understanding is highly beneficial, first, to obtain understanding in the biological design of cells and, second, as a possible basis for the building of artificial cortices for the stabilization of synthetic cells. Here, we report the results of coarse-grained molecular dynamics simulations of monomeric and filamentous actin in the vicinity of differently charged lipid bilayers. We observe that charges on the lipid head groups strongly determine the ability of actin to adsorb to the bilayer. The inclusion of divalent ions leads to a reversal of the binding affinity. Our in silico results are validated experimentally by reconstitution assays with actin on lipid bilayer membranes and provide a molecular-level understanding of the actin-membrane interaction.


Maternal soybean genistein on prevention of later-life breast cancer through inherited epigenetic regulations.
Breast cancer has strong developmental origins and maternal nutrition composition may influence later-life breast cancer risk in the offspring. Our study focused on a bioactive dietary component, genistein (GE) enriched in soybean products, to investigate specific timing of maternal GE exposure that may influence preventive efficacy of GE on offspring breast cancer later in life, and to explore the potential epigenetic mechanisms. Our results indicate a time-dependent effect of maternal GE exposure on early-life breast cancer development in offspring mice. Through integrated transcriptome and methylome analyses, we identified several candidate genes showing significantly differential gene expression and DNA methylation changes. We further found maternal long-term GE treatment can induce inherited epigenetic landmark changes in a candidate tumor suppressor gene, Trp63, resulting in transcriptional activation of Trp63 and induction of the downstream target genes. Our results suggest that maternal long-term exposure to soybean GE may influence early-life epigenetic reprogramming processes, which may contribute to its temporal preventive effects on breast cancer in the offspring. This study provides important mechanistic insights into an appropriate maternal administration of soybean products on prevention of breast cancer later in offspring life.


Synthesis, characterization and antimicrobial activity of water-soluble silver(i) complexes of metronidazole drug and selected counter-ions.
A series of water-soluble silver(i) complexes of the type [Ag(MTZ)2X] [MTZ = 1-(2-hydroxyethyl)-2-methyl-5-nitro-1H-imidazole (metronidazole drug); X = NO3(-), ClO4(-), CF3COO(-), BF4(-) and CH3SO3(-)] was synthesised by the reactions of various Ag(i) salts with metronidazole (MTZ). All the complexes were characterized by ESI-MS spectrometry, solution NMR ((1)H and (13)C) and IR spectroscopy, and elemental analysis. Further evidence for the formation and molecular structure of all the complexes was provided by X-ray single-crystal crystallography. The different counter ions affect the crystal packing of the complexes and thus have an impact on the final geometries. The antimicrobial activities of the complexes against two Gram-positive strains: Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, three Gram-negative strains: Pseudomonas aeruginosa ATCC 15442, Escherichia coli ATCC 25922, Proteus hauseri ATCC 13315 and yeast Candida albicans ATCC 10231 were evaluated and compared with antibacterial and antifungal properties of appropriate silver salts, metronidazole and silver sulfadiazine drugs. The newly synthesized compounds exhibited significant antibacterial activity against Gram-positive bacteria, better than the referenced silver sulfadiazine. The best active silver(i)-metronidazole complex contains a methanesulphonate counter-ion. Moreover, the complex inhibited the growth of yeast Candida albicans at a concentration 3-fold lower than that required for silver sulfadiazine. In addition, the complexes containing a tetrafluoroborate and a perchlorate as counter-ions were characterized as effective antibacterial agents against the tested Gram-negative bacteria.


[First Hungarian experience with pazopanib therapy for patients with metastatic renal cancer].
Pazopanib, a tyrosine kinase inhibitor, is one of the new registered first-line therapeutic options in the treatment of metastatic clear cell renal carcinoma. Our aim was to evaluate the efficiency and toxicity of first-line pazopanib therapy administered for patients with metastatic clear cell renal carcinoma with good- and medium prognosis according to MSKCC criteria. Between January and May, 2011, 24 patients have been treated with pazopanib in 8 oncology centers in Hungary, out of them 21 patients' data were analyzed. The mean age was 65.3 (49-81) years, 10 males and 11 females. According to MSKCC the prognosis was good and medium in 3 and 18 cases, respectively. Daily dose of pazopanib was 800 mg administered continuously in 28 day cycles. Dose reduction was performed according to the instructions of the registration study. Tumor response was evaluated according to RECIST 1.0. Currently 6 (28.6%) patients are on treatment. Dose reduction was necessary in 6 (28.6%) cases with an average duration of 14.55 (7-150) days. Mean±SE daily dose was 692.97±13.67 (400-800) mg. Median PFS was 12.41 months (95% CI 11.52-12.94 months). Complete remission (CR), as the best tumor response occurred in 2 (9.5%) cases. Partial remission (PR), stable disease (SD) and progression was observed in 6 (28.6%), 10 (47.6%) and 3 (14.3%) cases, respectively. Objective tumor response was observed in 8 pts (38%). Median survival could not be statistically analyzed yet due to the insignificant number of fatal outcomes. Median follow-up was 25.22 months (95% CI 2.47-28.1 months). As common side-effect fatigue, weakness and diarrhea occurred in 11 (52.4%), 9 (42.9%) and 8 (38%) cases, respectively. Besides these, worsening of high blood pressure and ALT/AST elevation was observed in 5 (23.8%) and 6 (28.6%) cases, respectively. Based on the initial Hungarian experiences, pazopanib is a well tolerable product and can be administered safely. According to our results its efficiency in terms of tumor response and progression-free survival is comparable to the results of the registration study.


Regulation of Nrf2 by Mitochondrial Reactive Oxygen Species in Physiology and Pathology.
Reactive oxygen species (ROS) are byproducts of aerobic respiration and signaling molecules that control various cellular functions. Nrf2 governs the gene expression of endogenous antioxidant synthesis and ROS-eliminating enzymes in response to various electrophilic compounds that inactivate the negative regulator Keap1. Accumulating evidence has shown that mitochondrial ROS (mtROS) activate Nrf2, often mediated by certain protein kinases, and induce the expression of antioxidant genes and genes involved in mitochondrial quality/quantity control. Mild physiological stress, such as caloric restriction and exercise, elicits beneficial effects through a process known as "mitohormesis." Exercise induces NOX4 expression in the heart, which activates Nrf2 and increases endurance capacity. Mice transiently depleted of SOD2 or overexpressing skeletal muscle-specific UCP1 exhibit Nrf2-mediated antioxidant gene expression and PGC1α-mediated mitochondrial biogenesis. ATF4 activation may induce a transcriptional program that enhances NADPH synthesis in the mitochondria and might cooperate with the Nrf2 antioxidant system. In response to severe oxidative stress, Nrf2 induces Klf9 expression, which represses mtROS-eliminating enzymes to enhance cell death. Nrf2 is inactivated in certain pathological conditions, such as diabetes, but Keap1 down-regulation or mtROS elimination rescues Nrf2 expression and improves the pathology. These reports aid us in understanding the roles of Nrf2 in pathophysiological alterations involving mtROS.


Structural characterization of fibrous synthetic hydrogels using fluorescence microscopy.
The structural features of the matrix surrounding the cells play a crucial role in regulating their behavior. Here, we used fluorescence microscopy and customized analysis algorithms to characterize the architecture of fibrous hydrogel networks. As a model system, we investigated a new class of synthetic biomimetic material, hydrogels prepared from polyisocyanides. Our results show that these synthetic gels present a highly heterogeneous fibrous network, with pores reaching a few micrometers in diameter. By encapsulating HeLa cells in different hydrogels, we show that a more porous structure is linked to a higher proliferation rate. The approach described here, for the characterization of the network of fibrous hydrogels, can be easily applied to other polymer-based materials and provide new insights into the influence of structural features in cell behavior. This knowledge is crucial to develop the next generation of biomimetic materials for 3D cell models and tissue engineering applications.


Analysis of cell survival after multiple fractions per day and low-dose-rate irradiation of two in vitro cultured rat tumor cell lines.
Two rat tumor cell lines which differ significantly in radiosensitivity, a rhabdomyosarcoma (R-1) and a ureter carcinoma (RUC-2), were treated with multiple fractions per day and low-dose-rate gamma radiation. The purpose of these experiments was to investigate (i) the influence of fraction size and interfraction interval on repair of sublethal damage (SD) and (ii) whether low-dose-rate irradiation can be simulated by giving multiple fractions per day which might be applied in clinical treatments. In both cell lines, multiple doses were given at 1- to 4-hr intervals. SD repair was at a maximum in 2 hr but did not reach the theoretically expected level. For both cell lines, survival at higher total doses was different from that theoretically expected if repair of SD was assumed to be completed and at the maximum level. To account for the observation that less than complete repair of SD occurred, theoretical survival curves were calculated with the assumption of a constant but less than 100% level of SD repair. Experimental data correlated well with these calculated curves. There were only very small differences in survival after the different multiple fractions per day regimens. Survival after irradiation at a dose rate of 1.00 Gy/hr was found to be similar to that after multiple fractions per day.


Intravenous allogeneic umbilical cord blood-derived mesenchymal stem cell therapy in recessive dystrophic epidermolysis bullosa patients.
BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) is an incurable disease that causes severe mucocutaneous fragility due to mutations in COL7A1 (encoding type VII collagen [C7]). In this phase I/IIa trial, we evaluated the safety and possible clinical efficacy of intravenous infusion of allogeneic human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in patients with RDEB.METHODSFour adult and two pediatric patients with RDEB were treated with 3 intravenous injections of hUCB-MSCs (1 × 106 to 3 × 106 cells/kg) every 2 weeks and followed up for 8-24 months after treatment. The primary endpoint was safety. Secondary endpoints related to efficacy included clinical parameters, such as disease severity score, wound assessment, itch and pain score, and quality of life. C7 expression levels and inflammatory infiltrates in the skin, as well as serum levels of inflammatory markers and neuropeptides, were also assessed.RESULTSIntravenous hUCB-MSC infusions were well tolerated, without serious adverse events. Improvements in the Birmingham Epidermolysis Bullosa Severity Score, body surface area involvement, blister counts, pain, pruritus, and quality of life were observed with maximal effects at 56-112 days after treatment. hUCB-MSC administration induced M2 macrophage polarization and reduced mast cell infiltration in RDEB skin. Serum levels of substance P were decreased after therapy. Increased C7 expression was observed at the dermoepidermal junction in 1 of 6 patients at day 56.CONCLUSIONTo the best of our knowledge, this is the first clinical trial of systemic administration of allogeneic hUCB-MSCs in patients with RDEB, demonstrating safety and transient clinical benefits.TRIAL REGISTRATIONClinicalTrials.gov NCT04520022.FUNDINGThis work was supported by Daewoong Pharmaceutical Co. Ltd. and Kangstem Biotech Co. Ltd.


Striatal mechanisms and pathogenesis of parkinsonian signs and motor complications.
Recent studies suggest that motor dysfunction associated with the chronic nonphysiologic stimulation of dopaminergic receptors on striatal spiny neurons alters the sensitivity of nearby glutamatergic receptors, especially those of the N-methyl-D-aspartate (NMDA) subtype. Lesioning the nigrostriatal dopamine system of rats or nonhuman primates induces parkinsonian signs; subsequent once- or twice-daily treatment with levodopa produces many of the features of the human motor complication syndrome. Some drugs that block NMDA receptors palliate parkinsonian signs in these animal models, as well as in patients with Parkinson's disease. Certain NMDA receptor antagonists injected into the striatum or given systemically also have the ability to act palliatively or prophylactically to alleviate levodopa-induced response alterations. These observations support the view that sensitization of striatal NMDA receptors contributes to the pathogenesis of motor dysfunction in Parkinson's disease. Since protein phosphorylation serves as an important regulatory mechanism for NMDA receptors, differential increases in the phosphorylation state of tyrosine and serine residues, observed as a result of nigrostriatal system destruction as well as response alteration induction, could account for the apparent augmentation in synaptic efficacy. Current evidence thus suggests that the intermittent stimulation of dopaminergic receptors activates signal transduction pathways in striatal neurons, which augment phosphorylation of NMDA receptors and thus their sensitivity to cortical glutamatergic input. As a result, striatal output changes in ways that favor the appearance of parkinsonian signs and motor complications.


Cloning and characterization of CLLD6, CLLD7, and CLLD8, novel candidate genes for leukemogenesis at chromosome 13q14, a region commonly deleted in B-cell chronic lymphocytic leukemia.
Chromosome 13q14 deletions constitute the most common structural aberration in B-cell chronic lymphocytic leukemia (B-CLL). We constructed a high-resolution physical map covering the critical deleted region in B-CLL at 13q14 and flanking sequences. The order and position of both genomic markers and known genes were determined precisely. Three novel genes, CLLD6, CLLD7, and CLLD8, were isolated and characterized. The predicted protein sequence of CLLD6 revealed no homology with known proteins. However, both CLLD7 and CLLD8 predicted proteins contain known functional domains. CLLD7 has both an RCC1 and a BTB domain, and could thus be involved in cell cycle regulation by chromatin remodeling. CLLD8 contains a methyl-CpG binding, a preSET and a SET domain, suggesting that CLLD8 might be associated with methylation-mediated transcriptional repression. Mutation analysis of hematopoietic tumor cell lines and B-CLL tumor samples revealed no point mutations within the coding region of these three novel genes. The functional domains present within CLLD7 and CLLD8 suggest that the proteins may be involved in critical cellular processes such as cell cycle and transcriptional control and could therefore be directly or indirectly involved in leukemogenesis.


Regulation of ribosomal S6 kinase 2 by mammalian target of rapamycin.
Phosphorylation of the ribosomal S6 subunit is tightly correlated with enhanced translation initiation of a subset of mRNAs that encodes components of the protein synthesis machinery, which is an important early event that controls mammalian cell growth and proliferation. The recently identified S6 kinase 2 (S6K2), together with its homologue S6K1, is likely responsible for the mitogen-stimulated phosphorylation of S6. Like S6K1, the activation of S6K2 requires signaling from both the phosphatidylinositol 3-kinase and the mammalian target of rapamycin (mTOR). Here we report the investigation of the mechanisms of S6K2 regulation by mTOR. We demonstrate that similar to S6K1 the serum activation of S6K2 in cells is dependent on mTOR kinase activity, amino acid sufficiency, and phosphatidic acid. Previously we have shown that mTOR is a cytoplasmic-nuclear shuttling protein. As a predominantly nuclear protein, S6K2 activation was facilitated by enhanced mTOR nuclear import with the tagging of an exogenous nuclear localization signal and diminished by enhanced mTOR nuclear export with the tagging of a nuclear export sequence. However, further increase of mTOR nuclear import by the tagging of four copies of nuclear localization signal resulted in its decreased ability to activate S6K2, suggesting that mTOR nuclear export may also be an integral part of the activation process. Consistently, the nuclear export inhibitor leptomycin B inhibited S6K2 activation. Taken together, our observations suggest a novel regulatory mechanism in which an optimal cytoplasmic-nuclear distribution or shuttling rate for mTOR is required for maximal activation of the nuclear S6K2.


Action of epidermal growth factor and retinoids on anchorage-dependent and -independent growth of nontransformed rat kidney cells.
Treatment of two nontransformed rat kidney cell lines with either retinoic acid or epidermal growth factor enhances anchorage-dependent cell growth and reduces cell substratum adhesiveness significantly, whereas the cell morphology is only moderately affected. Simultaneous treatment of these cells with these factors results in a dramatic change in cell morphology and increases cell growth and reduces cell substratum adhesiveness even further. Treatment of nontransformed rat kidney cells with certain retinoids enhances the binding of 125I-labeled mouse epidermal growth factor 2- to 3-fold. Addition of epidermal growth factor stimulates anchorage-independent growth of these cells. Retinoids enhance colony-forming ability in soft agar produced by epidermal growth factor, although retinoids by themselves do not affect anchorage-independent growth. Growth in soft agar induced by epidermal growth factor or epidermal growth factor plus retinoic acid treatments appears to be a reversible trait. Addition of epidermal growth factor stimulates secretion of plasminogen activator, whereas this production is not influenced by retinoids. A transformed rat kidney cell line, which exhibits very low epidermal growth factor binding and grows progressively in soft agar, is not significantly affected by retinoid treatment.


Patient-controlled analgesia compared with nurse-controlled infusion analgesia after heart surgery.
A randomized, controlled clinical trial was conducted on 66 patients undergoing elective cardiac surgery to compare patient-controlled analgesia (PCA) to nurse-controlled analgesia (NCA) with continuous morphine infusion. Hourly assessment of pain (at rest and on movement) using a visual analogue scale (VAS), of respiratory rate, and level of sedation took place for the 24 h following extubation. The incidence of nausea was also recorded. Mean pain scores were calculated, and peak pain and sedation scores, together with lowest respiratory rates, were identified. Morphine consumption was measured at 24 h. No significant differences were found between the groups' scores for pain or sedation. The PCA group had significantly lower respiratory rates (P = 0.02) and a lower incidence of nausea (P = 0.008). The PCA group also consumed significantly more morphine (P = 0.0001). The study suggests a beneficial effect from PCA after cardiac surgery in reducing nausea, compared to NCA. It confirms nurse-controlled infusion analgesia as an effective form of pain relief in an intensive care and high-dependency setting.


Single Nucleotide Polymorphisms in Cellular Drug Transporters Are Associated with Intolerance to Antiretroviral Therapy in Brazilian HIV-1 Positive Individuals.
Adverse reactions are the main cause of treatment discontinuation among HIV+ individuals. Genes related to drug absorption, distribution, metabolism and excretion (ADME) influence drug bioavailability and treatment response. We have investigated the association between single nucleotide polymorphisms (SNPs) in 29 ADME genes and intolerance to therapy in a case-control study including 764 individuals. Results showed that 15 SNPs were associated with intolerance to nucleoside and 11 to non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs), and 8 to protease inhibitors (PIs) containing regimens under alpha = 0.05. After Bonferroni adjustment, two associations remained statistically significant. SNP rs2712816, at SLCO2B1 was associated to intolerance to NRTIs (ORGA/AA = 2.37; p = 0.0001), while rs4148396, at ABCC2, conferred risk of intolerance to PIs containing regimens (ORCT/TT = 2.64; p = 0.00009). Accordingly, haplotypes carrying rs2712816A and rs4148396T alleles were also associated to risk of intolerance to NRTIs and PIs, respectively. Our data reinforce the role of drug transporters in response to HIV therapy and may contribute to a future development of personalized therapies.


Trends in Rheumatic Heart Disease-Related Mortality in the United States from 1999 to 2020.
There is a lack of mortality data on rheumatic heart disease (RHD) in the United States (US). In light of this, a retrospective analysis was conducted to investigate the temporal, sex-based, racial, and regional trends in RHD-related mortality in the US, ranging from 1999 to 2020. The Center for Disease Control and Prevention Wide-Ranging Online Data for Epidemiologic Research (CDC-WONDER) dataset was analyzed, where crude and age-adjusted mortality rates (AAMR) were identified, along with annual percentage changes (APCs) determined by Joinpoint regression. Through the period of 1999 to 2020, there were 141,137 RHD-related deaths reported, with a marginal decline from 4.05/100,000 in 1999 to 3.12/100,000 in 2020. However, the recent rise in AAMR from 2017 to 2020 has created a source of concern (APC: 6.62 [95% CI, 3.19-8.72]). Similar trends were observed in the Black or African American race from 2017 to 2020 (APC: 10.58 [95% CI, 6.29-17.80]). Moreover, the highest percentage change from 2018 to 2020 was observed in residents of large metropolitan areas (APC: 7.6 [95% CI, 2.8-10.5]). A prominent disparity was observed among states, with values ranging from 1.74/100,000 in Louisiana to 5.27/100,000 in Vermont. States within the top 90th percentile of RHD-related deaths included Alaska, Minnesota, Washington, Wyoming, and Vermont. In conclusion, it is imperative to delve deeper into the evidently rising trends of RHD-related mortality and outline the possible sources of social determinants within US healthcare in order to provide equal and quality medical care throughout the nation.


Nanofiber-expanded human umbilical cord blood-derived CD34(+) cell therapy accelerates cutaneous wound closure in NOD/SCID mice.
Nanofiber-expanded human umbilical cord blood-derived CD34(+) cell therapy has been shown to have potential applications for peripheral and myocardial ischaemic diseases. However, the efficacies of expanded CD34(+) cell therapy for treating cutaneous wounds and its mechanisms of action have yet to be established. Using an excisional wound model in non-obese diabetic/severe combined immune deficient mice, we show herein that CD34(+) cells accelerate the wound-healing process by enhancing collagen synthesis, and increasing fibroblast cell migration within the wound bed. Concomitantly, reduced levels of matrix metalloproteinase (MMPs) such as MMP1, MMP3, MMP9 and MMP13 were detected in the wound beds of animals treated with CD34(+) cells compared with vehicle-treated controls. CD34(+) cells were found to mediate enhanced migration and proliferation of dermal fibroblast cells in vitro. Moreover, CD34(+) cells secrete collagen in a serum-deprived environment. In mechanistic studies, co-culture of CD34(+) cells with primary skin fibroblasts increased the expression of collagen1A1, a component of type 1 collagen, and decreased the expression of MMP1 in fibroblast cells in the presence of a proteasome inhibitor. Finally, CD34(+) cell-mediated functions were transcriptionally regulated by the c-Jun N-terminal kinases pathway. Collectively, these data provide evidence of therapeutic efficacy and a novel mechanism of nanofiber-expanded CD34(+) cell-mediated accelerated wound healing.


Role of immune responses against the envelope and the core antigens of simian immunodeficiency virus SIVmne in protection against homologous cloned and uncloned virus challenge in Macaques.
We previously showed that envelope (gp160)-based vaccines, used in a live recombinant virus priming and subunit protein boosting regimen, protected macaques against intravenous and intrarectal challenges with the homologous simian immunodeficiency virus SIVmne clone E11S. However, the breadth of protection appears to be limited, since the vaccines were only partially effective against intravenous challenge by the uncloned SIVmne. To examine factors that could affect the breadth and the efficacy of this immunization approach, we studied (i) the effect of priming by recombinant vaccinia virus; (ii) the role of surface antigen gp130; and (iii) the role of core antigens (Gag and Pol) in eliciting protective immunity. Results indicate that (i) priming with recombinant vaccinia virus was more effective than subunit antigen in eliciting protective responses; (ii) while both gp130 and gp160 elicited similar levels of SIV-specific antibodies, gp130 was not as effective as gp160 in protection, indicating a possible role for the transmembrane protein in presenting functionally important epitopes; and (iii) although animals immunized with core antigens failed to generate any neutralizing antibody and were infected upon challenge, their virus load was 50- to 100-fold lower than that of the controls, suggesting the importance of cellular immunity or other core-specific immune responses in controlling acute infection. Complete protection against intravenous infection by the pathogenic uncloned SIVmne was achieved by immunization with both the envelope and the core antigens. These results indicate that immune responses to both antigens may contribute to protection and thus argue for the inclusion of multiple antigens in recombinant vaccine designs.


Endocrine-disrupting chemicals and uterine fibroids.
Uterine fibroids are the most frequent gynecologic tumor, affecting 70% to 80% of women over their lifetime. Although these tumors are benign, they can cause significant morbidity and may require invasive treatments such as myomectomy and hysterectomy. Many risk factors for these tumors have been identified, including environmental exposures to endocrine-disrupting chemicals (EDCs) such as genistein and diethylstilbestrol. Uterine development may be a particularly sensitive window to environmental exposures, as some perinatal EDC exposures have been shown to increase tumorigenesis in both rodent models and human epidemiologic studies. The mechanisms by which EDC exposures may increase tumorigenesis are still being elucidated, but epigenetic reprogramming of the developing uterus is an emerging hypothesis. Given the remarkably high incidence of uterine fibroids and their significant impact on women's health, understanding more about how prenatal exposures to EDCs (and other environmental agents) may increase fibroid risk could be key to developing prevention and treatment strategies in the future.


Dual Role of Hydrogen Peroxide as an Oxidant in Pneumococcal Pneumonia.
Significance:Streptococcus pneumoniae (Spn), a facultative anaerobic Gram-positive human pathogen with increasing rates of penicillin and macrolide resistance, is a major cause of lower respiratory tract infections worldwide. Pneumococci are a primary agent of severe pneumonia in children younger than 5 years and of community-acquired pneumonia in adults. A major defense mechanism toward Spn is the generation of reactive oxygen species, including hydrogen peroxide (H2O2), during the oxidative burst of neutrophils and macrophages. Paradoxically, Spn produces high endogenous levels of H2O2 as a strategy to promote colonization. Recent Advances: Pneumococci, which express neither catalase nor common regulators of peroxide stress resistance, have developed unique mechanisms to protect themselves from H2O2. Spn generates high levels of H2O2 as a strategy to promote colonization. Production of H2O2 moreover constitutes an important virulence phenotype and its cellular activities overlap and complement those of other virulence factors, such as pneumolysin, in modulating host immune responses and promoting organ injury. Critical Issues: This review examines the dual role of H2O2 in pneumococcal pneumonia, from the viewpoint of both the pathogen (defense mechanisms, lytic activity toward competing pathogens, and virulence) and the resulting host-response (inflammasome activation, endoplasmic reticulum stress, and damage to the alveolar-capillary barrier in the lungs). Future Directions: An understanding of the complexity of H2O2-mediated host-pathogen interactions is necessary to develop novel strategies that target these processes to enhance lung function during severe pneumonia.


Cardiovascular Complications of Hospitalized Patients With Coronavirus Disease 2019 in a Japanese Registry in 2020.
Cardiovascular complications of coronavirus disease 2019 (COVID-19) are critical for prognosis but have not been elucidated in Japan.Methods and Results:The COVID-19 Registry Japan, which included data from 19,853 individuals at the end of 2020, was analyzed. The incidences of cardiovascular complications were 0.098% for myocarditis/pericarditis/cardiomyopathy, 0.48% for ventricular tachycardia/fibrillation, 0.17% for myocardial ischemia, 0.062% for endocarditis, 0.59% for deep vein thrombosis, 0.19% for pulmonary embolism, and 0.37% for cerebral infarction/hemorrhage. Excluding endocarditis, all complications were associated with increased in-hospital mortality. Cardiovascular complications of COVID-19 were infrequent in Japan but were associated with poor prognosis.


The Role of the UPR Pathway in the Pathophysiology and Treatment of Bipolar Disorder.
Bipolar disorder (BD) is a mood disorder that affects millions worldwide and is associated with severe mood swings between mania and depression. The mood stabilizers valproate (VPA) and lithium (Li) are among the main drugs that are used to treat BD patients. However, these drugs are not effective for all patients and cause serious side effects. Therefore, better drugs are needed to treat BD patients. The main barrier to developing new drugs is the lack of knowledge about the therapeutic mechanism of currently available drugs. Several hypotheses have been proposed for the mechanism of action of mood stabilizers. However, it is still not known how they act to alleviate both mania and depression. The pathology of BD is characterized by mitochondrial dysfunction, oxidative stress, and abnormalities in calcium signaling. A deficiency in the unfolded protein response (UPR) pathway may be a shared mechanism that leads to these cellular dysfunctions. This is supported by reported abnormalities in the UPR pathway in lymphoblasts from BD patients. Additionally, studies have demonstrated that mood stabilizers alter the expression of several UPR target genes in mouse and human neuronal cells. In this review, we outline a new perspective wherein mood stabilizers exert their therapeutic mechanism by activating the UPR. Furthermore, we discuss UPR abnormalities in BD patients and suggest future research directions to resolve discrepancies in the literature.


Increased levels of dioxin-like substances in adipose tissue in patients with deep infiltrating endometriosis.
Are the levels of biologically active and the most toxic dioxin-like substances in adipose tissue of patients with deep infiltrating endometriosis (DIE) higher than in a control group without endometriosis? DIE patients have higher levels of dioxins and polychlorinated biphenyls (PCBs) in adipose tissue compared with controls without endometriosis. Some studies have investigated the levels of dioxin-like substances, in serum samples, in patients with endometriosis, with inconsistent results. Case-control study including two groups of patients. The study group (DIE group) consisted of 30 patients undergoing laparoscopic surgery because of DIE. In all patients, an extensive preoperative work-up was performed including clinical exploration, magnetic resonance imaging (MRI) and transvaginal sonography. All patients with DIE underwent a confirmatory histological study for DIE after surgery. The non-endometriosis control group (control group), included the next consecutive patient undergoing laparoscopic surgery in our center due to adnexal benign gynecological disease (ovarian or tubal procedures other than endometriosis) after each DIE patient, and who did not present any type of endometriosis. During the surgical procedure 1-2 g of adipose tissue from the omentum were obtained. Dioxin-like substances were analyzed in adipose tissue in DIE patients and controls without endometriosis. The total toxic equivalence and concentrations of both dioxins and PCBs were significantly higher in patients with DIE in comparison with the control group (P < 0.05), mainly due to the significantly higher values of the two most toxic dioxins (2,3,7,8-tetrachlorodibenzo-p-dioxin [2,3,7,8-TCDD] and 1,2,3,7,8-pentachlorodibenzo-p-dioxin [1,2,3,7,8-PeCDD]) (P < 0.01 for each compound). The levels of furan 2,3,4,7,8-PeCDF were statistically higher in the DIE group compared with controls. Only four congeners of PCBs had toxic equivalence values and concentrations that were statistically higher in patients with DIE, but these included the most toxic and carcinogenic PCB-126 (PCB-114 P < 0.05; PCB-156 P < 0.05; PCB-189 P = 0.04; PCB-126 P < 0.01). Since few patients were recruited, the study is only exploratory. Our results need to be confirmed in larger and more heterogeneous population studies since environmental and even genetic factors involved in determining dioxins and PCBs widely vary in different countries. Furthermore, the strict eligibility criteria used may preclude generalization of the results to other populations and the surgery-based sampling frame may induce a selection bias. Finally, adipose tissue was obtained only from the omentum, and not from other adipose tissue of the body. Our results suggest a potential role of dioxin-like substances in the pathogenesis of DIE. Further studies are warranted to confirm our findings. None. Not applicable.


Association of cinnarizine and betahistine in prophylactic therapy for Ménière's disease with and without migraine.
Prophylactic therapy of Ménière's disease (MD) includes betahistine and calcium-blockers (the latter also useful for migraine prevention). The aim of our work was to assess the efficacy of combined therapy with cinnarizine and betahistine in MD subjects both with and without migraine and poorly responsive to betahistine alone. Fifty-two MD subjects were included who were poorly responsive to betahistine during 6 months of follow-up; 29 were migraineurs. Combined therapy was administered with betahistine 48 mg/day and cinnarizine 20 mg BID for 1 month, 20 mg/day for 2 weeks and 20 mg every 2 days for 2 more weeks, and then repeated. Results were collected over 6 months of follow-up. MD subjects with and without migraine demonstrated a decrease in both vertigo spells and migrainous attacks during combined therapy (from 9.4 to 3.8 and from 6.8 to 5.9 in 6 months, respectively, for vertigo spells, while migraine decreased from 3.8 to 1 in 6 months, respectively). A correlation was seen between decrease of vertigo spells and headaches in the sample of MD subjects with migraine. Our data support a proactive role for cinnarizine in preventing vertigo spells, especially in MD patients with migraine.


Miscibility of acyl-chain defined phosphatidylcholines with N-palmitoyl sphingomyelin in bilayer membranes.
In this study we have used differential scanning calorimetry (DSC) to study the miscibility of different saturated phosphatidylcholines (PCs) with D-erythro-N-palmitoyl-sphingomyelin (16:0-SM). Information about the miscibility was obtained by observing the thermotropic phase behavior of binary mixtures of saturated PCs and 16:0-SM. The results obtained showed that PC miscibility in 16:0-SM was markedly affected by the PC acyl-chain composition. According to phase diagrams prepared from DSC data and the mid-transition temperatures of the main phase transition, the PC which formed the most ideal mixture with 16:0-SM was di-14:0-PC. However, the cooperativity of the main transition in PC/16:0-SM bilayers increased until the average acyl-chain length in the PC reached 15 carbons. Based on the criteria of the most ideal miscibility or the highest cooperativity of the main transition, we conclude that di-14:0-PC, 15:0/15:0-PC, and 14:0/16:0-PC interacted most favorably with 16:0-SM in bilayer membranes. Di-16:0-PC, to which 16:0-SM is often compared in biophysical studies, showed much less ideal miscibility.


Early acute antibody-mediated rejection of a negative flow crossmatch 3rd kidney transplant with exclusive disparity at HLA-DP.
Donor-specific alloantibodies (DSA) to HLA-DP may cause antibody-mediated rejection (AMR), especially in re-transplants. We describe the immunization history of a patient who received 3 kidney transplants; the 3rd kidney was completely matched except at DPA1 and DPB1. Prior to the 3rd transplant, single antigen bead analysis (SAB) showed DSA reactivity against DPA1 shared by the 1st and 3rd donors, but B and T flow crossmatch (FXM) results were negative. Within 11 days the 3rd transplant underwent acute C4d+ AMR which coincided with the presence of complement (C1q)-binding IgG1 DSA against donor DPA1 and DPB1. Using HLAMatchmaker and SAB, we provide evidence that eplet (epitope) spreading on DPA1 and eplet sharing on differing DPB1 alleles of the 1st and 3rd transplants was associated with AMR. Since weak DSA to DPA1/DPB1 may induce acute AMR with negative FXM, donor DPA1/DPB1 high resolution typing should be considered in sensitized patients with DP-directed DSA.


Terpene biosynthesis in glandular trichomes of hop.
Hop (Humulus lupulus L. Cannabaceae) is an economically important crop for the brewing industry, where it is used to impart flavor and aroma to beer, and has also drawn attention in recent years due to its potential pharmaceutical applications. Essential oils (mono- and sesquiterpenes), bitter acids (prenylated polyketides), and prenylflavonoids are the primary phytochemical components that account for these traits, and all accumulate at high concentrations in glandular trichomes of hop cones. To understand the molecular basis for terpene accumulation in hop trichomes, a trichome cDNA library was constructed and 9,816 cleansed expressed sequence tag (EST) sequences were obtained from random sequencing of 16,152 cDNA clones. The ESTs were assembled into 3,619 unigenes (1,101 contigs and 2,518 singletons). Putative functions were assigned to the unigenes based on their homology to annotated sequences in the GenBank database. Two mono- and two sesquiterpene synthases identified from the EST collection were expressed in Escherichia coli. Hop MONOTERPENE SYNTHASE2 formed the linear monterpene myrcene from geranyl pyrophosphate, whereas hop SESQUITERPENE SYNTHASE1 (HlSTS1) formed both caryophyllene and humulene from farnesyl pyrophosphate. Together, these enzymes account for the production of the major terpene constituents of the hop trichomes. HlSTS2 formed the minor sesquiterpene constituent germacrene A, which was converted to beta-elemene on chromatography at elevated temperature. We discuss potential functions for other genes expressed at high levels in developing hop trichomes.


p34cdc2 kinase activity is maintained upon activation of the replication checkpoint in Schizosaccharomyces pombe.
All eukaryotes use feedback controls to order and coordinate cell cycle events. In Schizosaccharomyces pombe, several classes of checkpoint genes serve to ensure that DNA replication is complete and free of error before the onset of mitosis. Wild-type cells normally arrest upon inhibition of DNA synthesis or in response to DNA damage, although the exact mechanisms controlling this arrest are unclear. Genetic evidence in fission yeast suggests that the dependence of mitosis upon completion of DNA replication is linked to the regulation of the p34cdc2 cyclin-dependent kinase. It has been hypothesized that inhibition of DNA synthesis triggers down-regulation of p34cdc2 kinase activity, although this has never been shown biochemically. We analyzed the activity of p34cdc2 in wild-type and checkpoint-defective cells treated with a DNA synthesis inhibitor. Using standard in vitro assays we demonstrate that p34cdc2 kinase activity is maintained in wild-type cells arrested at the replication checkpoint. We also used a novel in vivo assay for p34cdc2 kinase activity, in which we expressed a fragment of the human retinoblastoma tumor suppressor protein in fission yeast. Phosphorylation of this fragment of the human retinoblastoma tumor suppressor protein is dependent on p34cdc2 kinase activity, and this activity is also maintained in cells arrested at the replication checkpoint. These data suggest that the mechanism for cell-cycle arrest in response to incomplete DNA synthesis is not dependent on the attenuation of p34cdc2 activity.


Prospective Analysis of Cerebral Edema Admission and Clinical Outcome in Ruptured Intracranial Aneurysm.
To evaluate the association between global cerebral edema (GCE) after subarachnoid hemorrhage (SAH) and its impact on functional outcome evaluated by the modified Rankin scale (mRS). This is a prospective cohort study with patients who were admitted to the hospital due to SAH. During the period from January 2018 to November 2019, 107 patients with intracranial aneurysms were enrolled. Using univariate and multivariate analysis, we sought to identify predictors and evaluated the impact of GCE on outcome after 6 months using the mRS. GCE was present in 54 (50.5%) patients, of which 27 (25.2%) were mild, 20 (18.7%) moderate and 7 (6.5%) were severe. Univariate analysis identified high Hunt-Hess and Glasgow coma scale on clinical admission as predictors factors of GCE (p < 0.05), and higher modified Fisher scale as a radiological predictor of Glasgow coma scale (p < 0.05). Thirty-three (30.8%) patients were deceased at 6 months. Death or severe disability were predicted by higher age, poor clinical scale on admission and severe GCE (p < 0.05). GCE on admission is independently associated with poor clinical outcomes at discharge, and six months after SAH. Given its strong association with poor clinical grade on admission, GCE should be considered a straightforward and radiological important marker of early brain injury, with ominous implications.


Exome Variant Analysis of Chronic Periodontitis in 2 Large Cohort Studies.
Periodontitis is characterized by inflammation of the gingival tissue. The main risk factors are socioeconomic factors, sex, age, smoking, and diabetes, but periodontal disease has also a genetic background. Previous genome-wide association studies failed to reveal genome-wide significant associations of single common single-nucleotide polymorphisms with chronic periodontitis. Using the Illumina ExomeChip data of 6,576 participants of the German population-based cohort studies Study of Health in Pomerania (SHIP) and SHIP-Trend, the authors performed single variant and also gene-based association studies of rare and common exonic variations on different periodontal case definitions. Although our study comprised the largest sample size to date to assess genetic predisposition for chronic periodontitis, the authors found no significant association. This study emphasizes that for chronic periodontitis, large sample sizes will be necessary to find genetic associations, even when examining rare genetic variants.


Inhibition of spawning in zebrafish (Danio rerio): Adverse outcome pathways of quinacrine and ethinylestradiol.
This study determined the effects of the estrogen receptor agonist ethinylestradiol (EE2) and the phospholipase A2 inhibitor quinacrine (QUIN) on the pathways controlling follicular development, steroidogenesis, oocyte maturation, ovulation and spawning success in adult zebrafish. Both EE2 and QUIN inhibited spawning but did so through different mechanisms. EE2 affected follicular development (reduced ovarian size and reduction in the proportion of cortical alveolus, vitellogenic and mature follicle stages), steroidogenesis (reduced expression of aromatase), maturation (reduced luteinizing hormone receptor expression) and ovulation (reduced expression of cytosolic phospholipase A2 and the nuclear progesterone receptor). Although EE2 alters the proportion of follicle stages within the ovary, the downregulation of gene expression as a consequence of EE2 exposure was primarily due to a decline in expression of the genes of interest in vitellogenic and mature ovarian follicles. QUIN targeted ovulation via a reduction of the steroid 17α,20β dihydroxy-4-prenen-3-one (17α,20β-P) and decreased expression of the prostaglandin metabolizing enzyme cyclooxygenase 2. This study demonstrates the usefulness in defining the impacts of toxicants at the molecular and cellular, organ and whole organism level and how connections between these impacts can be used to describe the adverse outcome pathways (AOPs) that mediate toxicant action. Histological analysis and gene expression were effective tools in defining the AOPs of QUIN and EE2 while the measurement of reproductive hormones level did not provide much valuable information regarding the toxicant's mode of action.


Embolic and Other Adverse Outcomes in Symptomatic Versus Asymptomatic Patients With Atrial Fibrillation (from the ORBIT-AF Registry).
Asymptomatic atrial fibrillation (AF) is being increasingly diagnosed via implantable devices, screening, and inpatient telemetry. Management of asymptomatic AF is controversial, in part, because the associated risks have not been well described. We examined the incidence of major adverse outcomes in patients with asymptomatic versus symptomatic AF using Outcomes Registry for Better Informed Treatment of Atrial, a nationwide US registry of AF patients. We compared stroke and/or non-central nervous system (CNS) embolism, major adverse cardiovascular and neurologic events, bleeding, and death in 9,319 asymptomatic (defined by European Heart Rhythm Association score = 1 or "no symptoms") versus symptomatic patients. Overall, median (interquartile) age was 75 (67 to 82) years, 3,944 (42%) were women, and 38% versus 37% were asymptomatic based on physician versus patient-reported symptoms. Compared with those with symptoms, physician-defined asymptomatic patients were less likely to be woman (35%/47%) or be on an antiarrhythmic agent (22%/33%), but were more likely to have permanent and/or persistent AF (51%/40%). CHA2DS2-VASc scores did not vary by symptom status. After adjustment, risk of first stroke and/or non-CNS embolism (hazard ratio [HR] 0.85 [95% confidence interval {CI} 0.63 to 1.16], p = 0.32), major adverse cardiovascular and neurologic events (HR 0.88 [95% CI 0.76 to 1.03], p = 0.11), bleeding (HR 0.85 [95% CI 0.72 to 1.00], p = 0.05), and death (HR 0.99 [95% CI 0.87 to 1.13], p = 0.88) were similar in asymptomatic (European Heart Rhythm Association = 1) and symptomatic AF, respectively. Prospective, randomized studies are needed to further define associated adverse events and delineate optimal prophylactic therapies in patients with asymptomatic AF.


[Surgical correction of external nose deformities].
During surgical elimination of external nose deformation biologically inert prostheses were used. In the post-operative period low-intensity laser therapy was applied. No adverse effects of corundum ceramics were noted. The procedure of posterior tamponade was modified to ensure complete isolation of the nasopharynx during surgical manipulations on the external nose skeleton.


AAV2-mediated subretinal gene transfer of hIFN-α attenuates experimental autoimmune uveoretinitis in mice.
Recent reports show that gene therapy may provide a long-term, safe and effective intervention for human diseases. In this study, we investigated the effectiveness of adeno-associated virus 2 (AAV2) based human interferon-alpha (hIFN-α) gene therapy in experimental autoimmune uveoretinitis (EAU), a classic model for human uveitis. An AAV2 vector harboring the hIFN-α gene (AAV2.hIFN-α) was subretinally injected into B10RIII mice at two doses (1.5×10(6) vg, 1.5×10(8) vg). AAV2 vector encoding green fluorescent protein (AAV2.GFP) was used as a control (5×10(8) vg). The expression of hIFN-α in homogenized eyes and serum was detected by ELISA three weeks after injection. The biodistribution of vector DNA in the injected eyes, contralateral eyes and distant organs was determined by PCR. EAU was induced by immunization with IRBP(161-180) three weeks following vector injections, and evaluated clinically and pathologically. IRBP-specific proliferation and IL-17 expression of lymphocytes from the spleen and lymph nodes were assayed to test the influence of the subretinal delivery of AAV2.hIFN-α on the systemic immune response. hIFN-α was effectively expressed in the eyes from three weeks to three months following subretinal injection of AAV2.hIFN-α vector. DNA of AAV2.GFP was observed only in the injected eyes, but not in the distant organs or contralateral eyes. Subretinal injection of both doses significantly attenuated EAU activity clinically and histologically. For the lower dose, there was no difference concerning lymphocyte proliferation and IL-17 production among the AAV2.hIFN-α, AAV2.GFP and PBS injected mice. However, the higher dose of AAV2.hIFN-α significantly suppressed lymphocyte proliferation and IL-17 production. Subretinal delivery of AAV2.hIFN-α lead to an effective expression within the eye for at least three months and significantly attenuated EAU activity. AAV2.hIFN-α was shown to inhibit the systemic IRBP-specific immune response.


Internet of Things: Security and Solutions Survey.
The overwhelming acceptance and growing need for Internet of Things (IoT) products in each aspect of everyday living is creating a promising prospect for the involvement of humans, data, and procedures. The vast areas create opportunities from home to industry to make an automated lifecycle. Human life is involved in enormous applications such as intelligent transportation, intelligent healthcare, smart grid, smart city, etc. A thriving surface is created that can affect society, the economy, the environment, politics, and health through diverse security threats. Generally, IoT devices are susceptible to security breaches, and the development of industrial systems could pose devastating security vulnerabilities. To build a reliable security shield, the challenges encountered must be embraced. Therefore, this survey paper is primarily aimed to assist researchers by classifying attacks/vulnerabilities based on objects. The method of attacks and relevant countermeasures are provided for each kind of attack in this work. Case studies of the most important applications of the IoT are highlighted concerning security solutions. The survey of security solutions is not limited to traditional secret key-based cryptographic solutions, moreover physical unclonable functions (PUF)-based solutions and blockchain are illustrated. The pros and cons of each security solution are also discussed here. Furthermore, challenges and recommendations are presented in this work.


Tenascin-C: Friend or Foe in Lung Aging?
Lung aging is characterized by lung function impairment, ECM remodeling and airspace enlargement. Tenascin-C (TNC) is a large extracellular matrix (ECM) protein with paracrine and autocrine regulatory functions on cell migration, proliferation and differentiation. This matricellular protein is highly expressed during organogenesis and morphogenetic events like injury repair, inflammation or cancer. We previously showed that TNC deficiency affected lung development and pulmonary function, but little is known about its role during pulmonary aging. In order to answer this question, we characterized lung structure and physiology in 18 months old TNC-deficient and wild-type (WT) mice. Mice were mechanically ventilated with a basal and high tidal volume (HTV) ventilation protocol for functional analyses. Additional animals were used for histological, stereological and molecular biological analyses. We observed that old TNC-deficient mice exhibited larger lung volume, parenchymal volume, total airspace volume and septal surface area than WT, but similar mean linear intercept. This was accompanied by an increase in proliferation, but not apoptosis or autophagy markers expression throughout the lung parenchyma. Senescent cells were observed in epithelial cells of the conducting airways and in alveolar macrophages, but equally in both genotypes. Total collagen content was doubled in TNC KO lungs. However, basal and HTV ventilation revealed similar respiratory physiological parameters in both genotypes. Smooth muscle actin (α-SMA) analysis showed a faint increase in α-SMA positive cells in TNC-deficient lungs, but a marked increase in non-proliferative α-SMA + desmin + cells. Major TNC-related molecular pathways were not up- or down-regulated in TNC-deficient lungs as compared to WT; only minor changes in TLR4 and TGFβR3 mRNA expression were observed. In conclusion, TNC-deficient lungs at 18 months of age showed exaggerated features of the normal structural lung aging described to occur in mice between 12 and 18 months of age. Correlated to the increased pulmonary function parameters previously observed in young adult TNC-deficient lungs and described to occur in normal lung aging between 3 and 6 months of age, TNC might be an advantage in lung aging.


Current status of ramucirumab in gastroesophageal adenocarcinoma.
Outcomes of patients with advanced gastric or gastroesophageal junction adenocarcinoma (GEAC) remain poor despite recent advances. The standard of care in the management of this disease had not changed much over the past decade. In the first line, a platinum containing doublet/triplet is used, while in <20% of patients with human epithelial growth factor receptor type 2 overexpressing GEACs, trasuzumab can provide a modest advantage. Until recently, no standard second-line regimens existed; however, the results of the REGARD and RAINBOW trials led to the approval of ramucirumab in the second-line setting. From these trials it is clear that paclitaxel and ramucirumab should be used if possible. The placement of ramucirumab may become less clear as the data from immune oncology trials in GEAC emerge.


Motile cells as probes for characterizing acoustofluidic devices.
Acoustic microfluidics has emerged as a versatile solution for particle manipulation in medicine and biology. However, current technologies are largely confined to specialized research laboratories. The translation of acoustofluidics from research to clinical and industrial settings requires improved consistency and repeatability across different platforms. Performance comparisons will require straightforward experimental assessment tools that are not yet available. We introduce a method for characterizing acoustofluidic devices in real-time by exploiting the capacity of swimming microorganisms to respond to changes in their environment. The unicellular alga, Chlamydomonas reinhardtii, is used as an active probe to visualize the evolving acoustic pressure field within microfluidic channels and chambers. In contrast to more familiar mammalian cells, C. reinhardtii are simple to prepare and maintain, and exhibit a relatively uniform size distribution that more closely resembles calibration particles; however, unlike passive particles, these motile cells naturally fill complex chamber geometries and redistribute when the acoustic field changes or is turned off. In this way, C. reinhardtii cells offer greater flexibility than conventional polymer or glass calibration beads for in situ determination of device operating characteristics. To illustrate the technique, the varying spatial density and distribution of swimming cells are correlated to the acoustic potential to automatically locate device resonances within a specified frequency range. Peaks in the correlation coefficient of successive images not only identify the resonant frequencies for various geometries, but the peak shape can be related to the relative strength of the resonances. Qualitative mapping of the acoustic field strength with increasing voltage amplitude is also shown. Thus, we demonstrate that dynamically responsive C. reinhardtii enable real-time measurement and continuous monitoring of acoustofluidic device performance.


A case control study of lung cancer in Florence, Italy. I. Occupational risk factors.
A case control study of lung cancer in Florence, Italy was performed to investigate occupational risk factors for both men and women. The case series (n = 376) comprised all incident, histologically confirmed cases of primary lung cancer occurring in a three year period. Controls (n = 892) were patients in the same hospital of similar age, sex, date of admission, and smoking status with discharge diagnoses other than lung cancer or suicide. A detailed occupational history was collected from each subject directly. Logistic regression models were used to calculate odds ratios for specific occupations compared to all others. Among men, the lung cancer risk for bricklayers using firebrick and other refractory materials was elevated (adjusted odds ratio 6.5, 95% limits 2.1 and 20.9). Female hat makers, probably exposed to arsenic while making felt hats, had an elevated risk of lung cancer (6 cases versus 0 controls, p = 0.01). Risks in other occupations are discussed.


Molecular analysis of the human cytoglobin mRNA isoforms.
Multiple functions have been proposed for the ubiquitously expressed vertebrate globin cytoglobin (Cygb), including nitric oxide (NO) metabolism, lipid peroxidation/signalling, superoxide dismutase activity, reactive oxygen/nitrogen species (RONS) scavenging, regulation of blood pressure, antifibrosis, and both tumour suppressor and oncogenic effects. Since alternative splicing can expand the biological roles of a gene, we investigated whether this mechanism contributes to the functional diversity of Cygb. By mining of cDNA data and molecular analysis, we identified five alternative mRNA isoforms for the human CYGB gene (V-1 to V-5). Comprehensive RNA-seq analyses of public datasets from human tissues and cells confirmed that the canonical CYGB V-1 isoform is the primary CYGB transcript in the majority of analysed datasets. Interestingly, we revealed that isoform V-3 represented the predominant CYGB variant in hepatoblastoma (HB) cell lines and in the majority of analysed normal and HB liver tissues. CYGB V-3 mRNA is transcribed from an alternate upstream promoter and hypothetically encodes a N-terminally truncated CYGB protein, which is not recognized by some antibodies used in published studies. Little to no transcriptional evidence was found for the other CYGB isoforms. Comparative transcriptomics and flow cytometry on CYGB+/+ and gene-edited CYGB-/- HepG2 HB cells did not unveil a knockout phenotype and, thus, a potential function for CYGB V-3. Our study reveals that the CYGB gene is transcriptionally more complex than previously described as it expresses alternative mRNA isoforms of unknown function. Additional experimental data are needed to clarify the biological meaning of those alternative CYGB transcripts.


Prevalence of agglutinating antibodies to Toxoplasma gondii in striped skunks (Mephitis mephitis), opossums (Didelphis virginiana), and raccoons (Procyon lotor) from Connecticut.
The prevalence of agglutinating antibodies to Toxoplasma gondii was examined in striped skunks (Mephitis mephitis), opossums (Didelphis virginiana), and raccoons (Procyon lotor) from 8 cities in Connecticut. Ten (42%) of the 24 striped skunks, 2 of 7 (29%) opossums, and 12 of 12 (100%) raccoons were positive at dilutions of 1:50 or greater. These results suggest that T. gondii is prevalent in the environment, or prey items, or both, of these omnivores in Connecticut.


Properties of realgar bioleaching using an extremely acidophilic bacterium and its antitumor mechanism as an anticancer agent.
Realgar is a naturally occurring arsenic sulfide (or Xionghuang, in Chinese). It contains over 90% tetra-arsenic tetra-sulfide (As4S4). Currently, realgar has been confirmed the antitumor activities, both in vitro and in vivo, of realgar extracted using Acidithiobacillus ferrooxidans (A. ferrooxidans). Bioleaching, a new technology to greatly improve the use rate of arsenic extraction from realgar using bacteria, is a novel methodology that addressed a limitation of the traditional method for realgar preparation. The present systematic review reports on the research progress in realgar bioleaching and its antitumor mechanism as an anticancer agent. A total of 93 research articles that report on the biological activity of extracts from realgar using bacteria and its preparation were presented in this review. The realgar bioleaching solution (RBS) works by inducing apoptosis when it is used to treat tumor cells in vitro and in vivo. When it is used to treat animal model organisms in vivo, such as mice and Caenorhabditis elegans, tumor tissues grew more slowly, with mass necrosis. Meanwhile, the agent also showed obvious inhibition of tumor cell growth. Bioleaching technology greatly improves the utilization of realgar and is a novel methodology to improve the traditional method.


[Nonalcoholic steatohepatitis--a "new" hepatic disease].
Non-alcoholic fatty liver disease (NAFLD) is one of the most common hepatic disorders in the Western world. Non-alcoholic steatohepatitis (NASH) may occur in a subset of NAFLD patients and is an increasingly recognised clinicopathologial hepatic disorder. NASH may have significant impact on the healthcare system as it is associated with the metabolic syndrome comprising insulin resistance, obesity, hypertension, and type 2 diabetes mellitus. NASH can progress to liver fibrosis, cirrhosis and chronic hepatic failure and eventually to the need for a liver transplantation. The present review deals with the epidemiological features of NASH, describes a two-step pathogenesis with hepatic lipid accumulation (NAFLD) followed by the development of steatohepatitis (NASH). A strategy for establishing a diagnosis of NASH is presented including the indication for liver biopsy. The treatment of NASH may comprise different modalities from diet, weight loss, and exercise to pharmacological treatment to improve insulin resistance and drugs with antioxidant effects.


Murine low-density lipoprotein receptor-related protein 1 (LRP) is required for phagocytosis of targets bearing LRP ligands but is not required for C1q-triggered enhancement of phagocytosis.
C1q and members of the defense collagen family are pattern recognition molecules that bind to pathogens and apoptotic cells and trigger a rapid enhancement of phagocytic activity. Candidate phagocytic cell receptors responsible for the enhancement of phagocytosis by defense collagens have been proposed but not yet discerned. Engagement of phagocyte surface-associated calreticulin in complex with the large endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP/CD91), by defense collagens has been suggested as one mechanism governing enhanced ingestion of C1q-coated apoptotic cells. To investigate this possibility, macrophages were derived from transgenic mice genetically deficient in LRP resulting from tissue-specific loxP/Cre recombination. LRP-deficient macrophages were impaired in their ability to ingest beads coated with an LRP ligand when compared with LRP-expressing macrophages, confirming for the first time that LRP participates in phagocytosis. When LRP-deficient and -expressing macrophages were plated on C1q-coated slides, they demonstrated equivalently enhanced phagocytosis of sheep RBC suboptimally opsonized with IgG or complement, compared with cells plated on control protein. In addition, LRP-deficient and -expressing macrophages ingested equivalent numbers of apoptotic Jurkat cells in the presence and absence of serum. Both LRP-deficient and -expressing macrophages ingested fewer apoptotic cells when incubated in the presence of C1q-deficient serum compared with normal mouse serum, and the addition of purified C1q reconstituted uptake to control serum levels. These studies demonstrate a direct contribution of LRP to phagocytosis and indicate that LRP is not required for the C1q-triggered enhancement of phagocytosis, suggesting that other, still undefined, receptor(s) exist to mediate this important innate immune function.


Neonatal nicotine exposure impairs nicotinic enhancement of central auditory processing and auditory learning in adult rats.
Children of women who smoke cigarettes during pregnancy display cognitive deficits in the auditory-verbal domain. Clinical studies have implicated developmental exposure to nicotine, the main psychoactive ingredient of tobacco, as a probable cause of subsequent auditory deficits. To test for a causal link, we have developed an animal model to determine how neonatal nicotine exposure affects adult auditory function. In adult control rats, nicotine administered systemically (0.7 mg/kg, s.c.) enhanced the sensitivity to sound of neural responses recorded in primary auditory cortex. The effect was strongest in cortical layers 3 and 4, where there is a dense concentration of nicotinic acetylcholine receptors (nAChRs) that has been hypothesized to regulate thalamocortical inputs. In support of the hypothesis, microinjection into layer 4 of the nonspecific nAChR antagonist mecamylamine (10 microM) strongly reduced sound-evoked responses. In contrast to the effects of acute nicotine and mecamylamine in adult control animals, neither drug was as effective in adult animals that had been treated with 5 days of chronic nicotine exposure (CNE) shortly after birth. Neonatal CNE also impaired performance on an auditory-cued active avoidance task, while having little effect on basic auditory or motor functions. Thus, neonatal CNE impairs nicotinic regulation of cortical function, and auditory learning, in the adult. Our results provide evidence that developmental nicotine exposure is responsible for auditory-cognitive deficits in the offspring of women who smoke during pregnancy, and suggest a potential underlying mechanism, namely diminished function of cortical nAChRs.


Curcumin decreases the DNA adduct formation, arylamines N-acetyltransferase activity and gene expression in human colon tumor cells (colo 205).
The effects of curcumin on the N-acetyltransferase (NAT) activity, AF-DNA adduct formation and NAT gene expression were examined using the human colon tumor cell line (colo 205). Various concentrations of curcumin were added to the cytosols or to the medium of human colon tumor cells. The NAT activity was determined by high performance liquid chromatography assaying for the amounts of acetylated 2-aminofluorene (AAF) and p-aminobenzoic acid (N-Ac-PABA) and nonacetylated 2-aminofluorene (AF) and p-aminobenzoic acid (PABA). The NAT activity in the human colon tumor cells and cytosols was suppressed by curcumin in a dose-dependent manner. The results demonstrated that gene expression (NAT1 mRNA) in human colon tumor cells was inhibited by curcumin. The apparent values of Km and Vmax of NAT of human colon tumor cells were also decreased by curcumin in cytosols. Curcumin may act as a noncompetitive inhibitor. After the incubation of human colon tumor cells with AF with or without curcumin cotreatment, the cells were recovered and DNA was prepared, hydrolyzed to nucleotides, the adducted nucleotides were extracted into butanol and AF-DNA adducts analyzed by HPLC. The results also demonstrated that when curcumin was added to the media a decrease in AF-DNA adduct formation was seen in the human colon tumor cells. The finding of AF-DNA adduct formation in cultured human colon tumor cells suggests the usefulness of cultured cells for assessing arylamine-induced DNA damage.


Using relatedness networks to infer contemporary dispersal: Application to the endangered mammal Galemys pyrenaicus.
Information about the degree of contemporary dispersal is important when trying to understand how populations interchange individuals and identify the specific barriers that prevent these movements. In the case of endangered species, this can represent crucial information when designing appropriate conservation strategies. Here we analyse relatedness between individuals from different localities and use these data to infer whether dispersal occurred in recent generations. We applied this approach to the Pyrenean desman (Galemys pyrenaicus), a semiaquatic and endangered species endemic to the Iberian Peninsula. We studied this species in four primary rivers in the Iberian Range, where two ancient mitochondrial lineages are separated by a strict contact zone, suggesting the existence of complex dispersal patterns. Using next-generation sequencing, we obtained 912 SNPs from each specimen and estimated relatedness values between them. While relatedness networks were dense within each river, we found surprisingly few relationships between individuals from different rivers despite their close proximity in some cases, indicating much lower dispersal between rivers compared to dispersal within a single river. In agreement with this result, the degree of inbreeding was exceedingly high in most individuals. These data show that relatedness information can be crucial to understand the contemporary dispersal patterns and conservation status of specific populations of endangered species.


DAS181 Treatment of Severe Lower Respiratory Tract Parainfluenza Virus Infection in Immunocompromised Patients: A Phase 2 Randomized, Placebo-Controlled Study.
There are no antiviral therapies for parainfluenza virus (PIV) infections. DAS181, a sialidase fusion protein, has demonstrated activity in in vitro and in animal models of PIV. Adult immunocompromised patients diagnosed with PIV lower respiratory tract infection (LRTI) who required oxygen supplementation were randomized 2:1 to nebulized DAS181 (4.5 mg/day) or matching placebo for up to 10 days. Randomization was stratified by need for mechanical ventilation (MV) or supplemental oxygen (SO). The primary endpoint was the proportion of patients reaching clinical stability survival (CSS) defined as returning to room air (RTRA), normalization of vital signs for at least 24 hours, and survival up to day 45 from enrollment. A total of 111 patients were randomized to DAS181 (n = 74) or placebo (n = 37). CSS was achieved by 45.0% DAS181-treated patients in the SO stratum compared with 31.0% for placebo (P = .15), whereas patients on MV had no benefit from DAS181. The proportion of patients achieving RTRA was numerically higher for SO stratum DAS181 patients (51.7%) compared with placebo (34.5%) at day 28 (P = .17). In a post hoc analysis of solid organ transplant, hematopoietic cell transplantation within 1 year, or chemotherapy within 1 year, more SO stratum patients achieved RTRA on DAS181 (51.8%) compared with placebo (15.8%) by day 28 (P = .012). The primary endpoint was not met, but post hoc analysis of the RTRA component suggests DAS181 may have clinical activity in improving oxygenation in select severely immunocompromised patients with PIV LRTI who are not on mechanical ventilation. Clinical Trials Registration. NCT01644877.


The presence of blood in the transfer catheter negatively influences outcome at embryo transfer.
Embryo transfer (ET) influences pregnancy rates in patients undergoing assisted reproduction. Data are conflicting as to which variables affect ET success. This study examines variables that may affect outcome after ET in assisted reproductive technology patients who had high-quality embryos transferred. Over a 23 month period, 669 consecutive cycles were examined. Only patients having grade I and grade II embryos, or blastocyst transfers, were included in this retrospective analysis. A total of 584 consecutive cycles met study criteria. At the time of ET, the following variables were recorded: aborted first attempt at ET; presence of blood and/or mucus in or on the transfer catheter after ET; ease of ET as judged by provider; need for mock embryo transfer immediately before the actual transfer and retention of embryos in the transfer catheter. These variables were retrospectively analysed for their impact on implantation rate (IR) and clinical pregnancy rate (CPR). There were 290 gestations (49.7% CPR). Multiple attempts at ET, subjective difficulty of ET, performance of a sham pass immediately prior to embryo transfer, and presence of mucus on or in the catheter did not affect the CPR or IR. No difference was noted in the mean age of patients having or lacking any of these factors. There was a significant association between the presence of blood on or in the catheter and decreased IR (P = 0.015) and CPR (P = 0.004). Retained embryos also decreased IR (P = 0.03). Multivariable analysis confirmed that the presence of blood on the transfer catheter was the most important of these transfer characteristics in predicting IR (P = 0.042) and CPR (P = 0.018). These results suggest that when only high-grade embryos or blastocysts are transferred, the presence of blood on the catheter is associated with decreased IR and CPR in assisted reproduction.


Impact of a glycogen phosphorylase inhibitor and metformin on basal and glucagon-stimulated hepatic glucose flux in conscious dogs.
The effects of a glycogen phosphorylase inhibitor (GPI) and metformin (MT) on hepatic glucose fluxes (μmol · kg(-1) · min(-1)) in the presence of basal and 4-fold basal levels of plasma glucagon were investigated in 18-h fasted conscious dogs. Compared with the vehicle treatment, GPI infusion suppressed net hepatic glucose output (NHGO) completely (-3.8 ± 1.3 versus 9.9 ± 2.8) despite increased glucose 6-phosphate (G-6-P) neogenesis from gluconeogenic precursors (8.1 ± 1.1 versus 5.5 ± 1.1). MT infusion did not alter those parameters. In response to a 4-fold rise in plasma glucagon levels, in the vehicle group, plasma glucose levels were increased 2-fold, and NHGO was increased (43.9 ± 5.7 at 10 min and 22.7 ± 3.4 at steady state) without altering G-6-P neogenesis (3.7 ± 1.5 and 5.5 ± 0.5, respectively). In the GPI group, there was no increase in NHGO due to decreased glucose-6-phosphatase flux associated with reduced G-6-P concentration. A lower G-6-P concentration was the result of increased net glycogenesis without altering G-6-P neogenesis. In the MT group, the increment in NHGO (22.2 ± 4.4 at 10 min and 12.1 ± 3.6 at steady state) was approximately half of that of the vehicle group. The lesser NHGO was associated with reduced glucose-6-phosphatase flux but a rise in G-6-P concentration and only a small incorporation of plasma glucose into glycogen. In conclusion, the inhibition of glycogen phosphorylase a activity decreases basal and glucagon-induced NHGO via redirecting glucose 6-phosphate flux from glucose toward glycogen, and MT decreases glucagon-induced NHGO by inhibiting glucose-6-phosphatase flux and thereby reducing glycogen breakdown.


ISOLATION OF TRICHOMONAS VAGINALIS WITH PENICILLIN.
Exposure of Trichomonas vaginalis in vaginal discharge for 60 hours to 5,000-10,000 units of penicillin in 10.0 ml of a medium containing cysteine (0.15 per cent.), peptone, liver infusion, maltose and human serum was adequate to destroy the associated bacteria. Seven strains were isolated from seven women without a failure. This technic widens the field of investigation by offering a means of obtaining additional strains of bacteria-free Trichomonas vaginalis for comparative study. Efforts to isolate Trichomonas vaginalis by such laborious methods as washing, micropipetting and migration have all failed to yield bacteria-free cultures at this and other laboratories. The adherence of bacteria to the trichomonads, which appear to have sticky surfaces, and the relatively slow speed with which these protozoa swim are probably in large measure responsible for failure. Further trials by these methods now seem unnecessary. In the cases reported above the success of the penicillin method may be attributed to several factors. Resistance of Trichomonas vaginalis to penicillin in the concentrations reported above, the absence from the specimens of vaginal discharge of bacteria resistant to penicillin in these same concentrations, and the destruction of bacteria adherent to the sticky surfaces of the trichomonads perhaps played a large part in assuring successful isolation of the protozoa.


An in vivo 3D micro-CT evaluation of tooth movement after the application of different force magnitudes in rat molar.
To investigate the precise longitudinal change in the periodontal ligament (PDL) space width and three-dimensional tooth movement with continuous-force magnitudes in living rats. Using nickel-titanium closed-coil springs for 28 days, 10-, 25-, 50-, and 100-g mesial force was applied to the maxillary left first molars. Micro-CT was taken in the same rat at 0, 1, 2, 3, 10, 14, and 28 days. The width of the PDL was measured in the pressure and tension sides from 0 to 3 days. Angular and linear measurements were used to evaluate molar position at day 0, 10, 14, and 28. The finite element model (FEM) was constructed to evaluate the initial stress distribution, molar displacement, and center of rotation of the molar. The initial evaluation of PDL width showed no statistical differences among different force magnitudes. Tooth movement was registered 1 hour after force application and gradually increased with time. From day 10, greater tooth movement was observed when 10 g of force was applied. The FEM showed that the center of rotation in the molar is located in the center of five roots at the apical third of the molar roots. The rat's molar movement mainly consists of mesial tipping, extrusion of distal roots, intrusion of mesial root, palatal inclination, and mesial rotation. Although the initial tooth movement after the application of different force magnitudes until day 3 was not remarkably different, 10 g of force produced more tooth movement compared with heavier forces at day 28.


Chronic daily headache in children and adolescents: a 2-year prospective study.
Chronic daily headache in children and adolescents has not been well described. We analyzed data for 37 children and adolescents who presented with chronic daily headache to our Pediatric Headache Clinic over a 2-year period. These youngsters had five distinct headache patterns; 40% of them had the "comorbid" pattern, 35% were classified with new daily persistent headache, 15% with transformed migraine, 5% with chronic tension-type headache, and 5% could not be classified. There were no significant differences by diagnosis in externalizing and internalizing behaviors, type A behaviors, disability, pain severity, days missed from school, and number of coping skills employed. Children and adolescents with chronic daily headache have distinct clinical patterns, but for the most part, have similar disability. Differences between adult and childhood chronic daily headache are emphasized.