Internal transcribed spacer (ITS) sequence-based characterization of fungal isolates from multiple yogurt facilities-A case study.

Fungal spoilage remains a significant issue in dairy product quality, especially for cultured dairy products such as yogurt formulated without preservatives such as potassium sorbate. Fungal contamination can occur throughout the processing continuum, from the dairy farm environment to the finished product processing environment. As molecular characterization of fungal isolates is used more frequently, we obtained fungal isolates obtained in 2 yogurt processing facilities as part of routine fungal testing of raw materials (e.g., fruit preparations, added ingredients), in-process product samples, environmental samples (e.g., air plates, equipment surfaces such as valves, face plates, air nozzles), and finished product samples, to determine whether internal transcribed spacer (ITS) barcoding data would be helpful to support source tracking of fungal contamination issues. Internal transcribed spacer PCR amplification and sequencing allowed us to classify the 852 isolates from these 2 facilities into 200 unique ITS allelic types (AT), representing the phyla Ascomycota (743 isolates), Basidiomycota (97 isolates), and Mucoromycota (12 isolates). Thirty ITS AT were isolated from both facilities; 62 and 108 ITS AT were isolated from only facility A or only facility B, respectively. Nine ITS AT were each represented by more than 20 isolates; these AT comprised 53% of the 852 isolates. The considerable diversity of fungal isolates even within a single facility illustrates the challenge associated with controlling fungal contamination of dairy products. The ITS barcoding technique, however, did show promise for facilitating the source tracking of fungal contamination, particularly for ITS AT over-represented in a given facility. For example, we found evidence for equipment-specific reservoirs for 2 AT (14 and 219) in facility B. Our data suggest that despite its limited discriminatory power, ITS sequencing can provide initial information that can help trace fungal contamination along the processing continuum. However, development and implementation of discriminatory subtyping methods will be needed to further improve the ability to identify sources of fungal contamination in dairy facilities. Developing and implementing sampling plans that comprehensively capture yeast and mold diversity in a given processing facility remain a considerable challenge.

Evaluation of biopreservatives in Greek yogurt to inhibit yeast and mold spoilage and development of a yogurt spoilage predictive model.

Dairy products, including cultured dairy products such as cheese and yogurt, are susceptible to fungal spoilage. Traditionally, additives such as potassium sorbate have been used to control fungal spoilage; however, with consumer demand for clean-label products, other strategies to control fungal spoilage (e.g., biopreservatives) are increasingly being used in dairy formulations. In order to help the dairy industry better evaluate biopreservatives for control of fungal spoilage, we developed a challenge study protocol, which was applied to evaluate 2 commercially available protective cultures for their ability to control yeast and mold spoilage of Greek yogurt. Greek yogurt formulated with and without protective cultures was inoculated with a cocktail consisting of 5 yeasts and 1 mold to yield inoculum levels of 101 and 103 cfu/g of yogurt. The inoculated yogurts were stored at 7°C and fungal counts as well as time to visible growth, on the yogurt surface, of mycelium mold colonies or yeast were determined over shelf-life. Whereas fungal concentrations increased to spoilage levels (≥105 cfu/g) in all yogurt formulations at both inoculum levels by d 23 of storage at 7°C, no surface mold was observed over 76 d in any of the products formulated with protective cultures. Control yogurts without biopreservatives all showed surface mold by d 23. In order to allow industry to better evaluate the business effects of improved control of surface mold growth that can be achieved with protective cultures, we developed a Monte Carlo simulation model to estimate consumer exposure to visible mold growth in yogurt formulated without fungal inhibitors. Our model showed that initial mold contamination rate has the largest effect on the model outcome, indicating that accurate data on contamination rates are important for use of these models. When air plates were used, in a proof-of-concept approach, to estimate initial contamination rates in a small yogurt manufacturing operation, our model predicted that 550 ± 25.2 consumers (±standard deviation) would be exposed to visible mold growth for every 1 million cups of yogurt produced. With initial contamination rate data for individual facilities, this model could be used by industry to estimate the number of consumers exposed to visible mold spoilage and could allow industry to better assess the value of mold-control strategies.

Psychrotolerant spore-former growth characterization for the development of a dairy spoilage predictive model.

Psychrotolerant spore-forming bacteria represent a major challenge regarding microbial spoilage of fluid milk. These organisms can survive most conventional pasteurization regimens and subsequently germinate and grow to spoilage levels during refrigerated storage. To improve predictions of fluid milk shelf life and assess different approaches to control psychrotolerant spore-forming bacteria in the fluid milk production and processing continuum, we developed a predictive model of spoilage of fluid milk due to germination and growth of psychrotolerant spore-forming bacteria. We characterized 14 psychrotolerant spore-formers, representing the most common Bacillales subtypes isolated from raw and pasteurized milk, for ability to germinate from spores and grow in skim milk broth at 6°C. Complete growth curves were obtained by determining total bacterial count and spore count every 24 h for 30 d. Based on growth curves at 6°C, probability distributions of initial spore counts in bulk tank raw milk, and subtype frequency in bulk tank raw milk, a Monte Carlo simulation model was created to predict spoilage patterns in high temperature, short time-pasteurized fluid milk. Monte Carlo simulations predicted that 66% of half-gallons (1,900 mL) of high temperature, short time fluid milk would reach a cell density greater than 20,000 cfu/mL after 21 d of storage at 6°C, consistent with current spoilage patterns observed in commercial products. Our model also predicted that an intervention that reduces initial spore loads by 2.2 Log10 most probable number/mL (e.g., microfiltration) can extend fluid milk shelf life by 4 d (end of shelf life was defined here as the first day when the mean total bacterial count exceeded 20,000 cfu/mL). This study not only provides a baseline understanding of the growth rates of psychrotolerant spore-formers in fluid milk, it also provides a stochastic model of spoilage by these organisms over the shelf life of fluid milk, which will ultimately allow for the assessment of different approaches to reduce fluid milk spoilage.

Internal transcribed spacer (ITS) sequencing reveals considerable fungal diversity in dairy products.

Fungi are important spoilage organisms in dairy products. However, little is known about the diversity of naturally occurring spoilage fungi in raw milk and processed dairy products, due at least in part to the fact that classical fungal identification methods require considerable expertise. To gain further insight into the fungal diversity in the dairy system, we isolated fungi from raw milk, raw and pasteurized milk cheese, and yogurt using the selective dichloran rose bengal chloramphenicol agar. In total, 361 fungal isolates were obtained and further characterized by DNA sequencing of the internal transcribed spacer (ITS) region and the nuclear ribosomal large subunit (LSU) rRNA gene if needed. We conducted BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) searches of the ITS region sequences against the UNITE Database (https://unite.ut.ee/analysis.php), and selected other databases if needed, which allowed identification to the species level of 183 isolates and to the genus level of 107 of the 346 isolates that were successfully ITS sequenced. The isolates characterized represented 3 phyla and 19 genera; the most common genera isolated were Penicillium (25% of isolates), Debaryomyces (18%), and Candida (9%). This study not only provides, by using modern molecular tools, a baseline understanding of the types of fungi in dairy products, but also confirms that ITS sequencing is a useful approach for identification of fungal organisms found in the dairy food chain.

3T MRI reveals extra- and intracranial involvement in giant cell arteritis.

BACKGROUND AND PURPOSE: The frequency and amount of intracranial, intradural inflammatory vessel wall enhancement in giant cell arteritis remain unclear. The purpose of this work was to prospectively assess the intracranial extent of vasculitic changes in patients with giant cell arteritis using a dedicated MR imaging protocol optimized for assessment of mural changes of intracranial arteries. MATERIALS AND METHODS: Twenty-eight patients with suspected giant cell arteritis underwent 3T MR imaging. Imaging included a fat-saturated T1WI pre- and postcontrast application optimized for assessment of intradural vessel wall enhancement and high-resolution fat-saturated T1WI to evaluate superficial extracranial vessels. Temporal artery biopsies were available in 11 cases. Vessel wall enhancement of intradural and extracranial vessels was evaluated by 2 observers independently. RESULTS: Twenty patients had giant cell arteritis; 9 cases were biopsy-proved. Clear vessel wall enhancement of superficial extracranial and intradural internal carotid arteries was detected in 16 and 10 patients, respectively. Slight vessel wall enhancement of the vertebral arteries was seen. Of 9 patients with giant cell arteritis with vessel occlusion or stenosis, 2 presented with cerebral ischemic infarcts. Vessel occlusion or stenosis site coincided with the location of vessel wall enhancement of the vertebral arteries in 4 patients and of the intradural ICA in 1 patient. CONCLUSIONS: Vessel wall enhancement of intradural arteries, mainly the ICA, can be regularly found in patients with giant cell arteritis. Mural inflammatory changes of the intradural ICA detected on MR imaging may identify a subgroup of patients with giant cell arteritis and should be further evaluated in clinical studies.

Three-dimensional optoacoustic tomography at video rate.

Using optoacoustic excitation, a complete volumetric tomographic data sets from the imaged object can in principle be generated with a single interrogating laser pulse. Thus, optoacoustic imaging intrinsically has the potential for fast three-dimensional imaging. We have developed a system capable of acquiring volumetric optoacoustic data in real time and showcase in this work the undocumented capacity to generate high resolution three-dimensional optoacoustic images at a rate of 10 Hz, currently mainly limited by the pulse repetition rate of the excitation laser.

Do patients with invasive lobular breast cancer benefit in terms of adequate change in surgical therapy from a supplementary preoperative breast MRI?

BACKGROUND: Breast magnetic resonance imaging (MRI) has been introduced in the preoperative management of invasive lobular breast cancer (ILC). We analysed if MRI leads to adequate changes in surgical management. PATIENTS AND METHODS: We carried out a single-centre retrospective confirmatory analysis of 92 patients with ILC and a preoperative breast MRI. By applying a blinded tumour board method, we analysed if surgical procedures were altered due to breast MRI. In case of alteration, we analysed whether the change was adequate according to the postoperative pathology findings. We considered an adequate rate of change>5% to be a clinically relevant benefit. RESULTS: A change in surgical therapy due to the MRI findings occurred in 23 of 92 patients (25%). According to the postoperative pathology findings, this change was adequate for 20 of these patients (22%; 95% confidence interval [CI] 14%-31%, P<0.0001). An overtreatment occurred for three patients (3%; 95% CI 0%-6%) who underwent a mastectomy following the results of breast MRI. Patients with larger tumours did likely benefit more from preoperative breast MRI. CONCLUSIONS: Patients with ILC might benefit from a preoperative breast MRI. Possible harm from overtreatment should be minimised by diligent use of preoperative histological clarification.

Apple and peach consumption habits across European countries.

The aim of this study was to gain information concerning apple and peach consumption frequency within different European countries in relation to age and gender. The survey was a part of a complex experiment with the aim of evaluating consumers' preferences towards new varieties, and the data is based on the self-reported declarations of respondents, male and female, between 15 and 70 years old. 4271 consumers from 7 European countries were invited to supply information about their apple consumption habits, whereas 499 respondents from 5 countries answered questions relating to frequency of peach and nectarine consumption. In both, the apple and the peach surveys, data analysis of declared intake showed significant differences between nationalities. The highest apple consumption was in Poland, where over 55% declared a consumption of more than 5 apples per week. In comparison, Italian consumers most often indicated eating 3-5 apples per week (39.3%). The lowest apple consumption was in the Netherlands and Spain. In the case of peaches, the highest consumption was indicated in France where 48% of respondents declared a peach consumption of 3-5 per week with 40% eating more than 5 fruits per week. The lowest peach intake was declared in Germany. Irrespective of country women were shown to eat more apples that men. Furthermore, the group of older people (61-70 years) consume apples more often than the adult group (36-60), while within the youngest group of consumers (16-35) eating apples was not at all popular. As with apples females declared a higher peach consumption, and again significantly lower fruit consumption by the youngest group (16-35) was indicated. Although the availability of fruit at the market remains a prime factor in determining apple and peach consumption, our survey confirmed the trends of declining this popular fruit intake by the younger generation, as well as the persistent tendency of lower frequency of fruit consumption among men than women.

Social contexts in adolescent smoking: does school policy matter?

According to an ecological perspective in psychology and in line with social cognitive theory, smoking behaviour is determined by different social contexts (for example, peers, family and school) providing adolescents with important role models. This paper investigates the effects of personal characteristics as well as family, peer and school context variables on youth smoking behaviour. We hypothesize that school smoking policy variables predict adolescents' smoking in addition to other context variables. Data were obtained from a self-report survey administered to 3364 students in 40 secondary schools in Bavaria, Germany. For both younger (10-15 years) and older (16-21 years) students, strong associations were found between smoking behaviour and (i) smoking best friends and friends in general, (ii) other substance use and (iii) school performance. In the younger age group, the non-existence of smoking bans for students was associated with an increased risk of being smoker. For the older students, a positive association was found between the presence of smoking teachers on school grounds and smoking behaviour. Results are discussed considering methodological aspects and public health concerns.

Both long-term HIV infection and highly active antiretroviral therapy are independent risk factors for early carotid atherosclerosis.

OBJECTIVE: There is controversy over whether or not chronic HIV infection contributes to atherosclerosis. We investigated the relationship between HIV infection, antiretroviral medication and ultrasound evidence of early atherosclerosis in the context of vascular risk factors. DESIGN: A case-control design with 292 HIV-positive subjects and 1168 age- and sex-matched controls. METHODS: We assessed vascular risk factors, blood pressure, serum lipids and carotid intima media thickness (IMT) in cases and controls. With multivariate regression models, we investigated the effects of HIV status and antiretroviral medication on IMT. RESULTS: The common carotid artery (CCA) IMT value was 5.70% (95% confidence interval [3.08-8.38%], p<0.0001) or 0.044 mm [0.021-0.066 mm] (p=0.0001) higher in HIV-positives, adjusted for multiple risk factors. In the carotid bifurcation (BIF), the IMT values were 24.4% [19.5-29.4%] or 0.250 mm [0.198-0.303 mm] higher in HIV patients (p<0.0001). An investigation of antiretroviral substances revealed higher CCA- and BIF-IMT values in patients receiving combination antiretroviral therapy (HAART). CONCLUSIONS: HIV infection and HAART are independent risk factors for early carotid atherosclerosis. Assuming a risk ratio similar to that in large population-based cohorts, the observed IMT elevation suggests that vascular risk is 4-14% greater and the "vascular age" 4-5 years higher in HIV-positive subjects. The underlying mechanisms remain to be clarified.

Transgenic myocardial overexpression of fibroblast growth factor-1 increases coronary artery density and branching.

Fibroblast growth factor (FGF)-1 plays important roles during myocardial and coronary morphogenesis. FGF-1 is also involved in the physiological response of the adult heart against ischemia, which includes cardiomyocyte protection and vascular growth. In the present study, we have generated transgenic mice with specific myocardial overexpression of the gene. Transgene expression was verified by Northern blot, and increased FGF-1 protein content was assessed by Western blot and immunoconfocal microscopy. Anatomic, histomorphological, and ultrastructural analyses revealed no major morphological or developmental abnormalities of transgenic hearts. Capillary density was unaltered, whereas the density of coronary arteries, especially arterioles, was significantly increased, as was the number of branches of the main coronary arteries. In addition, the coronary flow was significantly enhanced in transgenic mice ex vivo. These differences in the anatomic pattern of the coronary vasculature are established during the second month of postnatal life. The present findings demonstrate an important role of FGF-1 in the differentiation and growth of the coronary system and suggest that it is a key regulatory molecule of the differentiation of the arterial system.

Controlled trial of transdermal nicotine patch in tobacco withdrawal.

A transdermal nicotine patch, which delivers 0.7 mg/cm2 per 24 h and is available in sizes of 10, 20, and 30 cm2 was tested in subjects from 21 general medical practices in a 3-month, placebo-controlled randomised double-blind study. The nicotine group (n = 100) and the placebo group (n = 99) were similar at entry. Participants who smoked more than 20 cigarettes a day were treated with the 30 cm2 patch and the others with the 20 cm2 patch. When abstinence, defined as smoking 0-3 cigarettes per week and verified by CO measurement, was achieved, the next smallest patch was applied. After 1, 2, and 3 months of treatment 41, 36, and 36%, respectively, in the nicotine group were abstinent. The corresponding figures in the placebo group were 19, 20, and 23%. The differences were significant for all 3 months. Body weight did not increase in the nicotine group, but in the placebo group the mean increase was 4.4 kg. Craving and withdrawal symptoms decreased more with nicotine substitution for cigarettes. The patches were generally well tolerated, although 25% of subjects in the nicotine group and 13% in the placebo group had transient local erythema after application of the patch; 5 members of the nicotine group withdrew because of poor cutaneous tolerance.

Mode and speed specificity of eccentric and concentric exercise training.

This research was supported by a Duke University Research Council Grant. The purpose of this study was to examine mode and speed specificity of strength training by comparing concentric and eccentric isokinetic exercise of the quadriceps. Forty-eight healthy men (mean age = 23.9 years) were randomly assigned to one of three groups: concentric training (C), eccentric training (E), or control (K). Average force (in Newtons) of 3 concentric and of 3 eccentric quadriceps contractions on the KIN-COM(R) dynamometer at 60, 120, and 180 degrees /sec was evaluated prior to and following a 6 week period during which only the C and E groups trained. Training sessions (3/week) included 4 submaximal and 1 maximal warm-up followed by 10 maximal effort isokinetic contractions of the quadriceps at 120 degrees /sec for each leg. Group C subjects trained concentrically only while Group E subjects trained eccentrically only. A t-test for independent means showed no significant right/left differences. ANOVA and Scheffe's F-tests were then used to assess the differences in training effects among the 3 groups for the left leg only. Results showed that although Group C increased slightly in both concentric and eccentric force at all speeds, the gains were significant only for concentric force at 180 degrees /sec. Group E showed significant gains (p < 0.05) in eccentric force at all speeds but not in concentric force. The K group had no significant change in concentric or eccentric force at any speed. We conclude that the eccentric mode of isokinetic exercise has highly specific strength training effects while the concentric mode has less specific training effects. In addition, speed of exercise does not appear to have specific training effects. J Orthop Sports Phys Ther 1989;11(2):70-75.

Experimental manipulations and marrow-derived factors which affect the outcome of bone marrow transplantation across the H-2 barrier in lethally irradiated mice.

A system is illustrated here for transplantation of bone marrow (BMT) across the H-2 barrier in conventionally raised mice. Timing of BM inoculation after supralethal irradiation, alteration of cellular composition of BM cell inoculum, sex of donor and recipient, inoculation or removal of BM-derived supernatants, use of BM-derived factors were all variables which profoundly affected survival, hemopoietic engraftment and induction of permanent, irreversible chimerism in different strains of mice (H-2d, H-2b, H-2k) transplanted with allogeneic BM. Inoculation of BM shortly after irradiation and addition or removal of plastic-adherent cells to the BM adversely affected survival and chimerism. Also inoculation of BM in its original supernatant containing all extracellular, endogenous BM factors resulted in increased mortality. BM-derived regulating factors (MRF) exerted variable effects in different H-2 combinations. However, heat-stable components of MRF greatly affected survival and chimerism. BM-derived supernatants and MRF from rabbit or different murine H-2 types thus seem to contain powerful inhibitors and promoters of BM engraftment. Promoters stimulate allogeneic BM engraftment, are heat-resistant and non-species specific. The different character and concentration of those factors between different H-2 types is also illustrated by their in vitro effects on BM cells. These results demonstrate that different experimental manipulations and factors which do not strictly depend on genetic diversity between donor and recipient profoundly affect allogeneic BMT. Adoption of manipulations combined with use of non-T cell depleted BM and the still largely unidentified BM-derived factors result in increment of survival and lasting chimerism without any manifestation of early or late graft versus-host disease (GVHD).

Transitory and persistent IgA deficiency. Reevaluation of 19 pediatric patients once found to be deficient in serum IGA.

In 21 out of 5181 serum samples tested between 1968 and 1977 IgA was not detectable. In this subsequent study, 19 individuals could be reinvestigated by clinical and laboratory methods. At reevaluation 26% showed high, 16% normal and 58% subnormal serum IgA. Late maturation of the IgA system therefore occurs frequently. Lack of secretory IgA was proven in 16% and the material used were tears: even in the group with subnormal IgA clinical manifestations are minimal. Development, compensatory mechanisms and criteria for a new definition of selective IgA deficiency are discussed.