Identification of Dairy Fungal Contamination and Reduction of Aflatoxin M1 Amount by Three Acid and Bile Resistant Probiotic Bacteria.

Aflatoxins (AFs) released by fungi are observed in the cow’s milk even after pasteurization. Aflatoxin M1 (AFM1) has particularly an incredible clinical significance, as a critical carcinogenic agent for humans. Several strategies have been implemented for lowering the AFM1 amount, such as the employment of probiotics, particularly lactobacilli or lactic acid bacteria (LAB). However, this strategy has not been applied routinely until today. This study aimed to evaluate the effect of three LABs on the reduction of AFM1 in traditional milk and cheese samples. In total, 85 milk (n=45) and cheese (n=40) samples were obtained from the open markets of Shiraz, Iran, from February to June 2018. Additionally, the AFM1 levels were evaluated, compared to those of the National Iranian Standard. The data were then analyzed in SPSS software (version 20) through the Chi-square test. Statistical analysis was performed at a 95% confidence level (p-value of <0.00001). Out of 50 purchased LABs, the efficient antifungal property and resistance to bile salts were observed in five strains. The mean value of these five strains was calculated after adding 5 ppm AFM1, compared to natamycin. The strains with a reduction in AFM1 level were sequenced and registered in the NCBI database.In total, 15 samples with contamination higher than the allowed limit included Penicillium spp, Aspergillus niger, Saccharomyces cerevisia, Saccharomyces paradoxus, and Yarrowia lipolytica.The results also showed reduced AFM1 levels in three LAB-treated strains. Lactobacillus fermentum CECT562 (T), Lactobacillus brevis ATCC14869 (T), and Enterococcus faecium LMG 11423 (T) had this capability to 0.05, 0.03, and 0.03 respectively. The National Iranian Standard should be implemented to have control over traditional dairy products with more care. The three LABs selected in the current study revealed a significant effect on reducing AFM1 levels in traditional milk and cheese.

Intracardiac electrogram envelope detection during atrial fibrillation using fast orthogonal search.

The performance of any intracardiac electrogram processing method is limited by the accuracy of its activation detection approach. The most common activation detection approaches in the literature aim to find the highest peak in the activation envelope disregarding the start and end points. However, the duration of the activation can be used to extract useful information such as wave collisions. In this work, we propose a novel orthogonal based approach for fast and accurate estimation of the start and end of the activations (activation envelope) in intracardiac recordings during atrial fibrillation. Wavelet decomposition of the signals was used to create a pool of basis functions for the proposed modeling method. The database included 24 recordings of approximate length of 6s obtained from atrial endocardium of 5 patients who underwent catheter ablation therapy. The start and end of activations in each electrogram was manually annotated by an expert electrophysiologist and the annotations were used as a gold standard to calculate the performance of our envelope detection method. The results show promising performance and excellent robustness to training data for our proposed method with respect to envelope estimation error.

Identification of Mucorales isolates from soil using morphological and molecular methods.

BACKGROUND AND PURPOSE: Soil is the main habitat of saprophytic and pathogenic fungi. Mucoromycotina constitutes a large group of soil fungi, with certain opportunistic members causing systemic infections in immunocompromised hosts. The majority of human and animal infections are caused by the members of the genera Rhizopus, Mucor, Rhizomucor, Lichtheimia (Absidia), Cunninghamella, and Mortierella. Accordingly, in the present study, we aimed to isolate and identify the main genera of the order Mucorales, using molecular assays and morphological features. MATERIALS AND METHODS: In total, 340 soil samples were collected from seven public parks throughout the city and sidewalk gardens in 14 municipal districts in Isfahan, Iran. All the samples were cultured on the appropriate media, incubated at 27°C for 2- 4 days, and examined daily for visible fungal growth. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was applied and macroscopic, microscopic, and physiological characteristics were assessed to identify fungal colonies. RESULTS: 400 pure colonies, belonging to the orders Mucorales and Mortierellales, including the genera Lichtheimia, Rhizopus, Rhizomucor, Mucor, Cunninghamella, and Mortierella, were identified. The genus Rhizopus (35.5%) was the most frequent isolate, followed by Mucor (32.25%) and Rhizomucor (27.5%). CONCLUSION: The results emphasize the importance of opportunistic fungi in public areas and indicate the risk of exposure for immunocompromised individuals.

Quantification of the role of tibial posterior slope in knee joint mechanics and ACL force in simulated gait.

The anterior cruciate ligament (ACL) rupture is a common knee joint injury with higher prevalence in female athletes. In search of contributing mechanisms, clinical imaging studies of ACL-injured individuals versus controls have found greater medial-lateral posterior tibial slope (PTS) in injured population irrespective of the sex and in females compared to males, with stronger evidence on the lateral plateau slope. To quantify these effects, we use a lower extremity musculoskeletal model including a detailed finite element (FE) model of the knee joint to compute the role of changes in medial and/or lateral PTS by ±5° and ±10° on knee joint biomechanics, in general, and ACL force, in particular, throughout the stance phase of gait. The model is driven by reported kinematics/kinetics of gait in asymptomatic subjects. Our predictions showed, at all stance periods, a substantial increase in the anterior tibial translation (ATT) and ACL force as PTS increased with reverse trends as PTS decreased. At mid-stance, for example, ACL force increased from 181 N to 317 N and 460 N as PTS increased by 5° and 10°, respectively, while dropped to 102 N and 0 N as PTS changed by -5° and -10°, respectively. These effects are caused primarily by change in PTS at the tibial plateau that carries a larger portion of joint contact force. Steeper PTS is a major risk factor, especially under activities with large compression, in markedly increasing ACL force and its vulnerability to injury. Rehabilitation and ACL injury prevention programs could benefit from these findings.

Steeper posterior tibial slope markedly increases ACL force in both active gait and passive knee joint under compression.

The role of the posterior tibial slope (PTS) in anterior cruciate ligament (ACL) risk of injury has been supported by many imaging studies but refuted by some in vitro works. The current investigation was carried out to compute the effect of ±5(o) change in PTS on knee joint biomechanics in general and ACL force/strain in particular. Two validated finite element (FE) models of the knee joint were employed; one active lower extremity musculoskeletal model including a complex FE model of the knee joint driven by in vivo kinematics/kinetics collected in gait of asymptomatic subjects, and the other its isolated unconstrained passive tibiofemoral (TF) joint considered under 1400 N compression at four different knee flexion angles (0°-45°). In the TF model, the compression force was applied at the joint mechanical balance point causing no rotations in sagittal and frontal planes. Changes in PTS moderately affected muscle forces and joint contact forces at mid-stance period. Both active (at mid-stance) and passive (at all flexion angles) models showed a substantial increase in the anterior tibial translation and ACL force as PTS increased with reverse trends as PTS decreased. In the active model of gait at mid-stance, ACL force increased by 75% (from 181 N to 317 N) in steeper PTS but decreased by 44% (to 102 N) in flatter PTS. The posterolateral bundle of ACL carried the load at smaller flexion angles with a shift to its anteromedial bundle as flexion increased. In accordance with earlier imaging studies, greater PTS is a major risk factor for ACL rupture especially in activities involving large compression forces.

Fibrodysplasia ossificans progressiva: report of a case and review of articles.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder, characterized by painful swelling of muscles and connective tissue in the early years of life, consequently leading to ossification at a mean age of 4-5 years. We report FOP in a 2-year-old boy with palpable masses in the frontal and lower cervical paraspinal and left periscapular muscles.He was born with hallux valgus. Despite this hallmark, he was referred to the hospital with the primary diagnosis of hematoma, but further investigation indicated FOP. The patient was discharged from the hospital with non steroidal anti-inflammatory drugs (NSAID) and education of the parents. The importance of this case was that in spite of the early occurrence of the typical presentation of FOP for more than one year and the fact that the patient's mother was a physician who had consulted with many specialists, the diagnosis had been missed.This indicates that the general physicians, radiologists and other specialists' awareness and knowledge of FOP is insufficient.

Study on Fungal Flora in the Midgut of the Larva and Adult of the Different Populations of the Malaria Vector Anopheles stephensi.

BACKGROUND: Many microorganisms in midgut of mosquito challenge with their host and also other pathogens present in midgut. The aim of this study was presence of non-pathogens microorganisms like fungal flora which may be crucial on interaction between vectors and pathogens. METHODS: Different populations of Anopheles stephensi were reared in insectary and objected to determine fungal flora in their midguts. The midgut paunch of mosquito adults and larvae as well as breading water and larval food samples transferred on Subaru-dextrose agar, in order to detect the environment fungus. RESULTS: Although four fungi, Aspergillus, Rhizopus, Geotrichum and Sacharomyces were found in the food and water, but only Aspiragilus observed in the midgut of larvae. No fungus was found in the midgut of adults. This is the first report on fungal flora in the midgut of the adults and larvae of An. stephensi and possible stadial transmission of fungi from immature stages to adults. CONCLUSION: The midgut environment of adults is not compatible for survivorship of fungi but the larval midgut may contain few fungi as a host or even pathogen.

Comparative study of coated and immobilized polysaccharide-based chiral stationary phases and their applicability in the resolution of enantiomers.

The enantioselective and chromatographic properties of Chiralpak AD and Chiralpak IA as well as those of Chiralcel OD and Chiralpak IB have been evaluated using a set of 48 compounds that differ in their physical and chemical properties. The impact of the different immobilisation methodologies of the chiral polysaccharide, i.e., coated or immobilized on retention and enantioselectivity was studied. The study on immobilized chiral stationary phases (CSPs) was expanded to also include mobile phases containing mixtures of alkanes and more non-conventional solvents such as ethyl acetate, ethers, acetone and dichloromethane. In this paper we report some of the general trends observed for the 48 racemic compounds with respect to retention, alpha and Rs. Further, the impact of the immobilisation methodology and the choice of the mobile phase on the elution order of the enantiomers is also discussed.

Methylation of the p16INK4A promoter is associated with malignant behavior in abdominal extra-adrenal paragangliomas but not pheochromocytomas.

Pheochromocytomas and abdominal extra-adrenal paragangliomas are related to endocrine tumors of the sympathetic nervous system. Studies in animal models have shown that inactivation of the products of the cyclin dependent kinase inhibitor 2A (CDKN2A) gene locus, p16INK4A and p14ARF, promotes the development of pheochromocytoma, especially in malignant form. The present study evaluated the involvement of CDKN2A in human pheochromocytomas and abdominal extra-adrenal paragangliomas from 55 patients. Promoter methylation was assessed using quantitative Pyrosequencing and methylation-specific PCR, and mRNA expression was measured by quantitative real-time PCR. For p16, western blot analysis and sequencing were also performed. succinate dehydrogenase complex subunit B (SDHB) sequencing analysis included extra-adrenal paragangliomas, all tumors classified as malignant, and cases diagnosed at 30 years or younger. The p16INK4A promoter was heavily methylated in a subset of paragangliomas, and this was significantly associated with malignancy (P<0.0043) and SDHB mutation (P<0.002). p16INK4A mRNA expression showed moderate suppression in malignant cases (P<0.05). In contrast, very little p14ARF promoter methylation was seen and there was no significant difference in p14ARF expression between tumors and normal samples. The p16 protein expression was reduced in 16 tumors, and sequence variations were observed in four tumors including the missense mutation A57V and the single nucleotide polymorphism (SNP) A148T. The results suggest that p16INK4A, and not p14ARF, is a subject of frequent involvement in these tumors. Importantly, hypermethylation of the p16INK4A promoter was significantly associated with malignancy and metastasis, and SDHB gene mutations. This finding suggests an etiological link and could provide a clinical utility for diagnostic purposes.

An alternative method of anthropometry of anterior cruciate ligament through 3-D digital image reconstruction.

Accurate and flexible measurements of length, area, and volume are important in evaluation of the mechanical properties of soft tissue. Although a number of contact-based and non-contact techniques have been reported in the literature, due to a variety of reasons such as cost, complexity, and low accuracy, the research community has not adopted a standardized technique. In this paper, an alternative method of measuring the geometric parameters of cadaver anterior cruciate ligament (ACL) is presented. In this method, a 3-D scan of the ACL is constructed using a simple, commercially available, scanning system. The 3-D scan is then analyzed using the 3-D Doctor Software to extract important information regarding the length, cross-sectional area, and volume of the ACL. The accuracy and repeatability of measurements obtained by this method are acceptable and comparable to existing non-contact methods. The limitation of the method is that surface concavities cannot be detected. However, the non-contact optical method, described here, has inherent advantages over the existing methods: (1) it is inexpensive; (2) it allows the determination of area at any distance along the length of the tissue of interest; (3) all relevant information including minimum area is extracted from one single application of the method; (4) the volume can be calculated with a simple additional step of length measurement although, for accurate results, condylar blockage must be minimized by coring the ACL out. The entire process of scanning takes less than 30 min. This technique has the potential to become a standard method in anthropometry of soft tissue.

Haplotype analysis and age estimation of the 113insR CDKN2A founder mutation in Swedish melanoma families.

Germline mutations in the CDKN2A tumor suppressor gene located on 9p21 have been linked to development of melanomas in some families. A germline 3-bp insertion in exon 2 of CDKN2A, leading to an extra arginine at codon 113 (113insR), has been identified in 17 Swedish melanoma families. Analysis of 10 microsatellite markers, spanning approximately 1 Mbp in the 9p21 region, showed that all families share a common allele for at least one of the markers closest to the CDKN2A gene, suggesting that the 113insR mutation is an ancestral founder mutation. Differences in the segregating haplotypes, due to meiotic recombinations and/or mutations in the short-tandem-repeat markers, were analyzed further to estimate the age of the mutation. Statistical analysis using a maximum likelihood approach indicated that the mutation arose 98 generations (90% confidence interval: 52-167 generations), or approximately 2,000 years, ago. Thus, 113insR would be expected to have a more widespread geographic distribution in European and North American regions with ancestral connections to Sweden. Alternatively, CDKN2A may lie in a recombination hot spot region, as suggested by the many meiotic recombinations in this narrow approximately 1-cM region on 9p21.

CDKN2A germ-line mutations in individuals with multiple cutaneous melanomas.

Germ-line CDKN2A mutations are present in some kindreds with hereditary cutaneous melanoma, and in Sweden a founder mutation with an extra arginine in codon 113 (113insR) has been identified. We screened 80 individuals with at least two primary cutaneous melanomas, who were identified mainly by a search of a regional cancer registry, for germ-line CDKN2A mutations. In nine patients, CDKN2A alterations that may contribute to melanoma predisposition were detected. In six individuals with a family history of melanoma, the 113insR founder mutation was present. One patient, who also had a family history of melanoma, had a 24-bp deletion that included codons 62-69. An in vitro binding assay established that the resulting mutant p16 protein was unable to bind cyclin-dependent kinase 4 and cyclin-dependent kinase 6. Two patients without a family history of melanoma had CDKN2A alterations: (a) one had a mutation in the 5' noncoding sequence (-14C/T); and (b) the other had an insertion of an extra T in codon 28, which results in a stop signal in codon 43. The median age at diagnosis of the first melanoma was significantly lower, the number of primary melanomas was significantly higher, and the presence of a family history of melanoma was significantly more common in patients with CDKN2A mutations than in those without germ-line mutations. The proportion of CDKN2A mutation carriers was significantly higher among patients treated for three or more primary melanomas compared with those with two tumors only. We conclude that mutation screening of individuals with multiple primary melanomas is a useful strategy to identify new melanoma kindreds with CDKN2A germ-line mutations.

Melanoma development in relation to non-functional p16/INK4A protein and dysplastic naevus syndrome in Swedish melanoma kindreds.

The CDKN2A gene encodes the cell cycle inhibitor p16/ INK4A, which is involved in familial cutaneous melanoma. We have studied five Swedish familial melanoma kindreds characterized by germline mutations in CDKN2A and dysplastic naevus syndrome (DNS). We found significant correlations between germline CDKN2A mutations and melanoma and between DNS phenotype and melanoma, respectively. There was also a correlation between mutation status and the presence of DNS. In CDKN2A mutation carriers, all cases of early-onset melanoma occurred in DNS individuals, and the mean age at melanoma diagnosis was significantly lower in individuals with DNS than in those without a confirmed DNS phenotype. In one family where the proband had a P48L mutation in CDKN2A exon 1, the DNS phenotype was studied in detail. In vitro binding experiments established that the P48L mutant protein does not bind to cdk4 or cdk6 and thus is functionally abnormal. Furthermore, we demonstrated loss of heterozygosity at markers on chromosome 9p flanking the CDKN2A locus in a primary melanoma and a metastasis from the proband. Our results are consistent with the hypothesis that germline CDKN2A mutations and DNS both contribute to the predisposition to melanoma and may lead to the development of early-onset melanoma when present in the same individual.

Analysis of mechanisms and frequency of CDKN2A/B gene loss during progression of RAS-transformed rat embryo fibroblast clones.

Rat embryo fibroblasts (REFs) are inefficiently transformed by RAS-oncogenes. Induction of p16INK4A expression by RAS has been suggested to contribute to this resistance. Glucocorticoid hormones, (DEX), enhance REF transformation by RAS and facilitates the isolation of transformed and immortal cell lines. We show that DEX induced cell proliferation is paralleled by a decrease in Cdkn2a gene transcripts, suggesting a mechanism for hormone promotion. The mechanisms of progression into hormone independent cell lines were examined. Twenty-two of 30 clones which reached a population size of approximately 10(6) cells could be established as cell lines. All lines studied showed homozygous deletions of the Cdkn2 loci (Cdkn2a and Cdkn2b) on RNO5. LOH was found for all RNO5 genetic markers examined in 7 of 19 cell lines, suggesting non-disjunction events. In the remaining 12 cell lines, both copies of Cdkn2 appeared to be lost by deletions/rearrangements, some of which could by demonstrated by karyotype analysis. We conclude that (i) clonal expansion of RAS-transfected REF by DEX is paralleled by down-regulation of Cdkn2a expression; (ii) homozygous deletion of Cdkn2 were estimated to occur at a frequency of 2 x 10(-8)/cell/generation or higher, and (iii) deletion/rearrangements and nondisjunction appear to be the main mechanisms leading to deletion of Cdkn2.

Four mutations in the porphobilinogen deaminase gene in patients with acute intermittent porphyria.

We have detected four different mutations in the porphobilinogen deaminase (PBGD) gene in acute intermittent porphyria (AIP) families from England, Norway, and Sweden. A splicing mutation in the first position of intron 8 (Int8 + 1) was found in a family from England and a missense mutation in exon 12 (Glu250) was detected in a Norwegian family. Two mutations were identified in Swedish families, one splicing mutation in the first position of intron 3 (Int3 + 1) and one missense mutation in exon 8 (Pro119).