Regulation of the clock gene expression in human adipose tissue by weight loss.

BACKGROUND: The circadian clock coordinates numerous metabolic processes to adapt physiological responses to light-dark and feeding regimens and is itself regulated by metabolic cues. The implication of the circadian clock in the regulation of energy balance and body weight is widely studied in rodents but not in humans. Here we investigated (1) whether the expression of clock genes in human adipose tissue is changed by weight loss and (2) whether these alterations are associated with metabolic parameters. SUBJECTS/METHODS: Subcutaneous adipose tissue (SAT) samples were collected before and after 8 weeks of weight loss on an 800 kcal per day hypocaloric diet (plus 200 g per day vegetables) at the same time of the day. Fifty overweight subjects who lost at least 8% weight after 8 weeks were selected for the study. The expression of 10 clock genes and key metabolic and inflammatory genes in adipose tissue was determined by quantitative real-time PCR. RESULTS: The expression of core clock genes PER2 and NR1D1 was increased after the weight loss. Correlations of PERIOD expression with body mass index (BMI) and serum total, high-density lipoprotein and low-density lipoprotein (LDL) cholesterol levels and of NR1D1 expression with total and LDL cholesterol were found that became non-significant after correction for multiple testing. Clock gene expression levels and their weight loss-induced changes tightly correlated with each other and with genes involved in fat metabolism (FASN, CPT1A, LPL, PPARG, PGC1A, ADIPOQ), energy metabolism (SIRT1), autophagy (LC3A, LC3B) and inflammatory response (NFKB1, NFKBIA, NLRP3, EMR1). CONCLUSION: Clock gene expression in human SAT is regulated by body weight changes and associated with BMI, serum cholesterol levels and the expression of metabolic and inflammatory genes. Our data confirm the tight crosstalk between molecular clock and metabolic and inflammatory pathways involved in adapting adipose tissue metabolism to changes of the energy intake in humans.

Craniofacial and dental development in cardio-facio-cutaneous syndrome: the importance of Ras signaling homeostasis.

Cardio-facio-cutaneous syndrome (CFC) is a RASopathy that is characterized by craniofacial, dermatologic, gastrointestinal, ocular, cardiac, and neurologic anomalies. CFC is caused by activating mutations in the Ras/mitogen-activated protein kinase (MAPK) signaling pathway that is downstream of receptor tyrosine kinase (RTK) signaling. RTK signaling is known to play a central role in craniofacial and dental development, but to date, no studies have systematically examined individuals with CFC to define key craniofacial and dental features. To fill this critical gap in our knowledge, we evaluated the craniofacial and dental phenotype of a large cohort (n = 32) of CFC individuals who attended the 2009 and 2011 CFC International Family Conferences. We quantified common craniofacial features in CFC which include macrocephaly, bitemporal narrowing, convex facial profile, and hypoplastic supraorbital ridges. In addition, there is a characteristic dental phenotype in CFC syndrome that includes malocclusion with open bite, posterior crossbite, and a high-arched palate. This thorough evaluation of the craniofacial and dental phenotype in CFC individuals provides a step forward in our understanding of the role of RTK/MAPK signaling in human craniofacial development and will aid clinicians who treat patients with CFC.

Effects of plant species richness on invasion dynamics, disease outbreaks, insect abundances and diversity.

Declining biodiversity represents one of the most dramatic and irreversible aspects of anthropogenic global change, yet the ecological implications of this change are poorly understood. Recent studies have shown that biodiversity loss of basal species, such as autotrophs or plants, affects fundamental ecosystem processes such as nutrient dynamics and autotrophic production. Ecological theory predicts that changes induced by the loss of biodiversity at the base of an ecosystem should impact the entire system. Here we show that experimental reductions in grassland plant richness increase ecosystem vulnerability to invasions by plant species, enhance the spread of plant fungal diseases, and alter the richness and structure of insect communities. These results suggest that the loss of basal species may have profound effects on the integrity and functioning of ecosystems.

Freezing of cells--replacement of serum by oxypolygelatine.

A commercially available plasma expander (Gelifundol) containing 5.5% oxypolygelatine in buffered saline was used instead of serum as a supplement for freezing several types of human cell and a mouse myeloma cell line in liquid nitrogen. Viability, recovery, proliferation and cytotoxic activity were compared after freezing with a plasma expander and after conventional freezing with human AB serum or fetal calf serum. The plasma expander proved to be equivalent or superior to AB serum by all parameters tested and acceptable even when compared with fetal calf serum. Furthermore, this preparation is cheap, sterile, free of BSE, viral and mycoplasmal contamination or antibodies and foreign serum proteins. We therefore recommend it for freezing of cells for culture of HLA typing.

HLA class I and class II antibodies: monitoring before and after kidney transplantation and their clinical relevance.

BACKGROUND: In search of an alternative screening technique, we compared complement-dependent cytotoxicity (CDC) with PRA-STAT, a commercially available enzyme-linked immunosorbent assay (ELISA). METHODS: A total of 188 pre- and posttransplant sera from 50 renal allograft recipients were tested with both methods. RESULTS: A significant correlation was found between both methods. Discrepant results could be explained by the fact that PRA-STAT detects both HLA class I and II antibodies (while CDC with peripheral blood lymphocytes as target cell detects mainly HLA class I reactivity), by the presence of IgM antibodies (which are not detected by the IgG-specific ELISA test), and by CDC "false-positive" results due to antibody rejection treatment. The clinical relevance of antibodies detected by PRA-STAT is suggested by the following. (a) In eight patients, donor-specific HLA antibodies detected by PRA-STAT (but not seen by CDC) resulted in severe rejection episodes, which led to graft loss in four cases. In all but one patient, antibodies were directed against class II or mixtures of class I and H antigens. Six patients with complications were shown to have developed de novo antibodies against DQ incompatibilities. (b) Half of the patients with a positive ELISA test at the moment of crossmatch experienced complications. Such patients are at a threefold higher risk of suffering from rejection episodes and/or graft loss than patients who are not sensitized (P<0.05, Fisher exact test). CONCLUSIONS: Because PRA-STAT is very reproducible, detects both HLA class I and II antibodies, and is not influenced by rejection therapy, we consider it an additional tool for pre- and posttransplant monitoring of kidney allograft recipients.

The association of kidney graft outcome with pretransplant serum IgG-anti-F(ab')2 gamma activity.

Pretransplant sera of 474 kidney graft recipients were tested for IgG-anti-F(ab')2 gamma activity. The patients had significantly higher IgG-anti-F(ab')2 gamma activity than healthy controls (P = 0.0004). Serum lymphocytotoxic antibodies were correlated with IgG-anti-F(ab')2 gamma (P = 0.004), whereas CMV infection and blood transfusions were not. We found a significant association between pretransplant IgG-anti-F(ab')2 gamma activity and early and 1-year kidney graft outcome. This association was pronounced in recipients with no lymphocytotoxic antibodies. Recipients with immediately functioning grafts and a creatinine < 130 mumol/L at 1 year had strikingly higher pretransplant IgG-anti-F(ab')2 gamma activity than patients with graft failure (P < 0.0001).

Role of protein tyrosine kinase on regulation of trabecular meshwork and ciliary muscle contractility.

PURPOSE: Trabecular meshwork and ciliary muscle express properties of smooth muscle cells. The contractility of trabecular meshwork and ciliary muscle is differently modulated by various agents. To reveal contractile regulatory processes, the effects of activation and inhibition of protein tyrosine kinases (PTKs) and their interaction with other protein kinases on contractility were measured. METHODS: Measurements of isometric tension were performed on isolated bovine trabecular meshwork and ciliary muscle strips using a custom-built, electromagnetic, force-length transducer. Protein tyrosine kinase (PTK) was stimulated by epidermal growth factor (EGF) and was inhibited by genistein or tyrphostin 51. Protein kinase C (PKC) was inhibited by chelerythrine or NPC-15437 and protein kinases A and G (PKA-PKG) by H8. RESULTS: Isolated strips were precontracted by applying carbachol 10(-6) M for 30 minutes (100% carbachol maximum contraction). Inhibition of PTK evoked a maximum relaxation of 79.2+/-4.2% in trabecular meshwork and of 38.1+/-3.1% in ciliary muscle (n=8). Inhibition of PKC or PKA-PKG induced relaxations only in trabecular meshwork. When PTK and PKC or PKA-PKG were inhibited, the relaxation induced by inhibition of PTK was additive to inhibition of the other protein kinases. Stimulation of a receptor with PTK activity by EGF induced a relaxation in trabecular meshwork and a contraction in ciliary muscle precontracted by carbachol. When trabecular meshwork and ciliary muscle were activated by EGF, inhibition of PTK by genistein relaxed the cell preparations. CONCLUSIONS: Inhibition of PTK induces more prominent relaxation in trabecular meshwork than in ciliary muscle. The effects of inhibition of PTK on relaxation are independent of inhibition of PKC and PKA-PKG. The signaling cascade after activation of a tyrosine kinase receptor by EGF is differently modulated in trabecular meshwork and ciliary muscle. The effect of genistein on relaxation is probably not directly related to the EGF receptor. PTK inhibitors are possible agents for the development of novel antiglaucoma drugs.

Reduced immunogenicity of long-term, passively enhanced rat renal allografts and passive transfer of graft protection.

Enhancing alloantiserum was produced by immunizing male BD IX inbred rats with density gradient separated nylon-wool adherent spleen lymphocytes from male rats of the major histocompatibility complex (MHC) different inbred strain Sprague-Dawley (SD). Long-term surviving (BD IX X SD) F1 to BD IX kidney grafts were achieved by treating the recipients with 1 ml alloantiserum at the time of transplantation. After greater than 100 days 7 passively enhanced F1 kidneys were retransplanted into naive BD IX rats. Four out of 7 secondary recipients, producing only low levels of lymphocytotoxic antibodies, survived for greater than 100 days, 3/7 rats died of surgical or infection complications. Twenty-one naive BD IX recipients of normal F1 kidneys were treated with serum (1 ml i.v.) and/or spleen lymphocytes (1 X 10(7) i.v.) obtained from the long-term survivors. An indefinite graft survival was achieved in 13 out of 21 rats. After greater than 150 days 6 out of these 13 passively enhanced F1 kidneys were retransplanted into naive BD IX rats which were challenged at the time of grafting with 4 X 10(7) SD lymphocytes to elicit rejection. Six out of 6 kidneys survived greater than 150 days. Thus, the long-term survival of rat kidney allografts in this model is associated with a strong reduction of graft immunogenicity as well as the development of suppressor cells and enhancing antibodies.

Variability of repeated coronary artery calcium measurements by electron beam tomography.

In 120 patients, the mean interscan variability of coronary calcium quantification by electron beam tomography was 19.9% (median 7.8%) for the traditional calcium score, and 16.2% (median 5.7%) for volumetric scoring. Although this difference was not significant, there was a significant influence of the total amount of calcium, number of acquired images, and image noise on interscan reproducibility.

Dynamics of donor-reactive IgG, IgA and IgM antibodies against T and B lymphocytes early after clinical kidney transplantation using flow cytometry.

Using flow cytometry, 32 kidney graft recipients were monitored retrospectively for at least 1 month to study the dynamics of serum IgG, IgA and IgM antibodies against donor T and B lymphocytes before and after transplantation. Donor spleen lymphocytes were used as targets. In the B cell cross-match, the surface immunoglobulins were blocked with an anti-human immunoglobulin antibody. A high frequency of donor-reactive antibodies was detected early after transplantation, especially when the sera were tested against B lymphocytes. Surprisingly, donor-reactive antibodies of the IgA isotype made up a substantial proportion of all antibodies detected. Within the first month after transplantation, six out of 32 patients (19%) developed IgG antibodies against donor T lymphocytes and nine out of 35 patients (28%) formed IgG antibodies against B lymphocytes. A similar situation was found for IgA antibodies: 22% of the recipients were positive for IgA antibodies against donor T lymphocytes and 34% against B lymphocytes after transplantation. Lower antibody frequencies were found for IgM antibodies (16% and 19%, respectively). From our data we conclude that for at least some of the parameters studied their individual dynamics reflect the complex immunological mechanisms occurring early after transplantation.

Development of Met-enkephalin immunoreactivity in organotypic explants of fetal mouse spinal cord and attached dorsal root ganglia.

Radioimmunoassays of methionine-enkephalin (Met-Enk) in organotypic cultures of 13-day fetal mouse spinal cord explants with attached dorsal root ganglia (DRG) demonstrate a progressive development of immunoreactivity (IR) during 5 weeks in vitro. Met-Enk IR in these cultures increased to levels observed in adult rodent spinal cord. most of the Met-Enk IR assays were made on cord explants excised from cord-DRG cultures. In smaller numbers of assays performed on entire DRG-cord cultures or on cord cultured in the absence of DRGs, similar levels of Met-Enk IR were obtained. Thus most of the Met-Enk IR appeared to be located within the cord tissue. No Met-Enk IR was detected in DRGs cultured in the absence of cord. In contrast, low levels of Met-Enk IR were present in about 50% of the assays of DRGs cultured attached to the cord. Since these assays included the neuritic outgrowths of the cultures, our data do not preclude possible contamination by Met-Enk immunoreactive cord neurites that may have aberrantly projected into the outgrowth zones. Nevertheless, the data raise the possibility of a trophic influence of cord tissue on the development of Met-Enk IR in DRG neurons. The development of Met-Enk IR in cord regions of cord-DRG explants extends previous binding assays demonstrating development of opiate receptors in these cultures and provides further support to electrophysiological analyses suggesting tonic opioid inhibitory networks in these explants.

Nonrandom Distribution of Virulences Within Two Field Collections of Uromyces appendiculatus.

ABSTRACT Two collections of urediniospores of Uromyces appendiculatus, each from a different commercial bean field, were characterized for associations of virulence among individuals within each collection. Four bean (Phaseolus vulgaris) lines with distinct, race-specific resistance to which virulence in each population was polymorphic were used to obtain measures of all six possible pairwise virulence associations for each collection. We inoculated one of the lines and collected urediniospores only from the segment of the population that was virulent on that line. This segment, when compared with nonselected collections from susceptible Pinto 111, gave a direct measure of degree of association as the change in frequency of virulence observed. Plants of the second bean line were inoculated in separate sets with both selected and unselected collections. Frequencies of virulence were estimated from the numbers of susceptible-type and resistant-type infections. Reciprocals of each pairing also were made. For collection P21, all virulences were significantly associated, either positively or negatively, except one pair (in one direction of selection only); whereas, for collection M5, all virulences were significantly associated. Virulence association in P21 was shown to be the result of predominance of phenotypes with certain combinations of virulence by inoculation of the four bean lines with 10 randomly chosen single-uredinial individuals. In support of this, a large random-mated F1 population derived from each collection showed much less virulence association, with the majority of pairs of virulences showing nonsignificant changes in virulence frequency after passage through the first line. Random mating also significantly changed virulence frequency from that of the original population in all instances. Changes were in both directions, suggesting either that virulences were not all recessive, or that heterozygote frequency was sometimes above and sometimes below the Hardy-Weinberg expectation in the field populations.

Comparison of Virulence and Isozyme Phenotypes of Pgt-QCCJ and Great Plains Races of Puccinia graminis f. sp. tritici.

ABSTRACT Stem rust race Pgt-QCCJ was first found in the Great Plains of the United States in 1989, collected primarily from barley. This race became a major part of the Puccinia graminis f. sp. tritici population, even though it is virulent to only a few hard red winter wheat cultivars in the central Great Plains and to barley in the northern Great Plains. It threatens barley production in the northern Great Plains of the United States and Canada due to virulence to Rpg-1. Six differences in virulence and two in isozyme banding patterns from the most similar stem rust races make it unlikely that QCCJ arose as a mutant. Thus, QCCJ likely arose through sexual or parasexual recombination. Sexual recombination in the Great Plains is unlikely, as it has not been detected in many years. Avirulence to 'McNair 70l' is only known from the Pacific Northwest of the United States and adjacent Canada. The rust population in this area is of sexual origin, and the pattern of virulence/avirulence and isozyme banding for QCCJ occurs there. Pgt-QCCJ likely originated in the Pacific Northwest during or before 1989 and was wind-transported into the Great Plains.

Variation at the b Mating Type Locus of Ustilago maydis.

ABSTRACT Population level diversity at the Ustilago maydis b mating type locus was determined in samples from four Minnesota locations using a combination of plate mating techniques and a polymerase chain reaction (PCR)-based assay. The PCR method allows rapid identification of b types from samples of natural populations and utilizes the hypervariable regions of the b locus that determine mating type specificity. Results demonstrated high levels of b diversity within populations, with one population yielding 17 of the total 18 b types found in the study. Pairwise G(ST) values were in the range of 0.02 to 0.05, and common b mating types were found across broad geographic distances. These data demonstrated that very low levels of differentiation among U. maydis populations occur with respect to b locus variation. Consistent with frequency-dependent selection models, b types were represented at approximately equal frequencies within the entire Minnesota population. However, neutral evolutionary models for patterns of geographic distribution and variation at b cannot be entirely excluded. The importance to agricultural practices of understanding population genetic processes is discussed.

Intensified ESWL of gallstones: dissociation of pulverisation, pain relief and stone-clearance.

BACKGROUND/AIMS: Recently, intensified shock wave lithotripsy for gallstone pulverisation and subsequent clearance without bile salt medication has been advocated. We report our first 44 patients treated by this regime: Patients with intact gallbladder function and symptomatic gallstones of any size, number and composition. METHODOLOGY: Forty-four consecutive patients who received intensified shock wave lithotripsy for gallstone pulverisation and clearance were included in this study. The patients all had intact gallbladder function and presented with symptomatic gallstones of any size, number and composition. RESULTS: Pulverisation was achieved in 75% of all cases (12 months), but only 34% were stone free. The proportion of patients with stone pulverisation compared to subsequent complete clearance was 93% versus 60% in small (

Repeatability and Relationship of Incidence and Severity Measures of Scab of Wheat Caused by Fusarium graminearum in Inoculated Nurseries.

Breeders of hard red spring wheat (Triticum aestivum) are attempting to incorporate resistance to scab, caused by Fusarium graminearum. In artificially inoculated, replicated field plots, 37 wheat entries (inbred lines or cultivars) were evaluated for 3 years and an additional 60 entries for 2 of the 3 years for incidence (percent spikes infected), severity (percent infected spikelets within infected spikes), and disease index (percent infected spikelets in 50-spike sample). From year to year, entries had similar index values, with coefficients of determination (r 2) ranging from 0.59 to 0.78, with a mean of 0.73. Entries appeared slightly more similar from year to year for incidence than for severity, although both measures of disease had highly significant r 2 values. Incidence and severity were highly correlated in the wheat germ plasm examined; r 2 values in single years ranged from 0.51 to 0.67, with a mean of 0.64. A representative subset of 22 entries was included for a fourth year. None of the measures of disease in year 4 correlated with their counterparts in any of the first 3 years. This loss of repeatability may have been caused by severe lodging or by high temperatures during the evaluation period that accelerated disease progress and wheat maturity during year 4. Incidence and severity remained correlated in year 4 (r 2 = 0.60).

Evaluation of a pro-active strategy for managing tuberculosis-HIV co-infection in a UK tertiary care setting.

Management of tuberculosis (TB)-HIV co-infection is complicated by interactions between the diseases and their therapies. We developed and evaluated a strategy to (i) treat co-infected patients in a single co-infection clinic, (ii) maximize use of first-line drugs, (iii) delay antiretroviral therapy (ART) until two months post-TB treatment except in severe immunosuppression, (iv) commence efavirenz at 600 mg daily with therapeutic drug monitoring (TDM) and (v) target treatment completion. We conducted a prospective cohort review over 5.5 years in a UK tertiary referral center where 56 HIV-positive patients treated for TB were followed-up for a median 30 months. Main outcome measures were treatment completion, adverse events, immune reconstitution inflammatory syndrome, immunological and virological parameters, and TDM for efavirenz. Treatment completion rates were 88% (49/56); four patients were lost to local follow-up and three (5.4%) died during treatment; no deaths were TB-related. Adverse events were common (55%), but caused no treatment interruptions. Standard doses (600 mg daily) of efavirenz with rifampicin achieved or exceeded therapeutic levels in 25/28 (89%). This study supports combined management for TB-HIV co-infected patients. Delaying ART to two months post-TB treatment did not seem to result in poor clinical outcomes in this well-resourced context. Although efavirenz 600 mg daily usually achieved satisfactory levels, TDM is recommended.