Physical and chemical changes in Alhydrogel™ damaged by freezing.

Accidental freezing of aluminum-based vaccines occurs during their storage and transportation, in both developed and developing countries. Freezing damages the freeze-sensitive aluminum adjuvanted vaccines, through separation of lattice between aluminum adjuvant and antigen, leading to formation of aluminum aggregates, and loss of potency. In this study, we examined Alhydrogel™ ([AlO(OH)]xnH2O, aluminum hydroxide, hydrated for adsorption) stored under recommended conditions, and exposed to freezing temperature until solid-frozen. The main purpose of our research was to determine the destruction areas of the solid-frozen Alhydrogel™ using selected methods of scanning electron microscopy, energy dispersive X-ray spectroscopy, Raman spectroscopy, Fourier-transform infrared spectroscopy and transmission electron microscopy working in diffraction mode. The Zeta potential evaluation, measurements of albumin adsorption power, thermogravimetric analysis and estimation of the mass loss after drying indicated significant structural (physical) and chemical differences between the freeze-damaged and non-frozen vaccine adjuvant. The presented results are important to better understand the type and nature of damages occurring in freeze-damaged aluminum-based vaccines. These results can be used in future studies to improve the temperature stability of aluminum adjuvanted vaccines.

Cytogenomic Evaluation of Children with Congenital Anomalies: Critical Implications for Diagnostic Testing and Genetic Counseling.

Identification of submicroscopic chromosomal aberrations, as a cause of structural malformations, is currently performed by MLPA (multiplex ligation-dependent probe amplification) or array CGH (array comparative genomic hybridization) techniques. The aim of this study was the evaluation of diagnostic usefulness of MLPA and array CGH in patients with congenital malformations or abnormalities (at least one major or minor birth defect, including dysmorphism) with or without intellectual disability or developmental delay and the optimization of genetic counseling in the context of the results obtained. The MLPA and array CGH were performed in 91 patients diagnosed with developmental disorders and major or minor congenital anomalies. A total of 49 MLPA tests toward common microdeletion syndromes, 42 MLPA tests for subtelomeric regions of chromosomes, two tests for common aberrations in autism, and five array CGH tests were performed. Eight (9 %) patients were diagnosed with microdeletion MLPA, four (4 %) patients with subtelomeric MLPA, one (1 %) patient with autism MLPA. Further three (3 %) individuals had rearrangements diagnosed by array CGH. Altogether, chromosomal microaberrations were found in 16 patients (17 %). All the MLPA-detected rearrangements were found to be pathogenic, but none detected with array CGH could unequivocally be interpreted as pathogenic. In patients with congenital anomalies, the application of MLPA and array CGH techniques is efficient in detecting syndromic and unique microrearrangements. Consistent pre-MLPA test phenotyping leads to better post-test genetic counseling. Incomplete penetrance and unknown inheritance of detected variants are major issues in clinical interpretation of array CGH data.

Pexophagy in Penicillin G Secretion by Penicillium chrysogenum PQ-96.

Penicillin G oversecretion by Penicillium chrysogenum PQ-96 is associated with a strictly adjusted cellular organization of the mature and senescent mycelial cells. Abundant vacuolar phagy and extended cellular vacuolization combined with vacuolar budding resulting in the formation of vacuolar vesicles that fuse with the cell membrane are the most important characteristic features of those cells. We suggest as follows: if the peroxisomes are integrated into vacuoles, the penicillin G formed in peroxisomes might be transferred to vacuoles and later secreted out of the cells by an exocytosis process. The peroxisomal cells of the mycelium are privileged in penicillin G secretion.

Quantitative expression of Candida albicans aspartyl proteinase genes SAP7, SAP8, SAP9, SAP10 in human serum in vitro.

The different members of the secreted aspartyl proteinase (Sap) family of the human pathogenic yeast Candida albicans are proposed to play different roles during infection and are differentially expressed at various body sites. In recent reports, expression analysis has focused on the genes SAP1-6, while the expression pattern of SAP7-10 was less well studied. We analyzed the SAP7-SAP10 expression profile of C. albicans under human serum influence that may be elucidated in the course of blood infection in humans and how this in vitro expression profile is associated with hyphal formation. The phenotypes of strains were examined under scanning electron microscopy. Quantitative RT-PCR (2-(deltadeltaC)T) was used to monitor SAP expression of C. albicans wild type cells and mutants lacking SAP9 and/or SAP10. Of the four analyzed SAP genes, only SAP7 was detectably induced in the double mutant and in the wild type strains in the model that mimics bloodstream infections. On the other hand, in the wild types (isolate 83 and CAF2-1), SAP7 was expressed 0.8- or 0.4-fold less than SAP10, respectively. Our findings suggest that Sap7 may respond to the challenge of the human blood environment. Furthermore, the results support the notion that compensatory upregulation of SAP7 and SAP8 in the deltasap9/deltasap10 mutant occurs in these conditions. SAP7-10 expression was strain-specific. Our findings point to a link between morphogenesis and expression of SAP9 in serum, where these conditions induce both hyphae and SAP9, but temporal gene expression patterns might be controlled by other factors.

Compartmentalization in penicillin G biosynthesis by Penicillium chrysogenum PQ-96.

The arrangement of organelles in the sub-apical productive non-growing vacuolated hyphal cells of the high- and the low-penicillin-pro- ducing strains Penicillium chrysogenum was compared using transmission electron microscopy. In the productive cells of the high-yielding strain the endoplasmic reticulum and the polyribosomes with associated peroxisomes are frequently arranged at the periphery of the cytoplasm and around the vacuoles. At the high activity of penicillin G biosynthesis the immuno-label of the cytosolic isopenicillin N synthase is concentrated at the polyribosomes arranged in the peripheral cytoplasm and along the tonoplast as well as around the peroxisomes. On the basis of the obtained results the compartmentalization of the pathway of penicillin G biosymthesis is discussed. The obtained results support the phenylacetic acid detoxification hypothesis of penicillin G biosynthesis.

Antifungal agents commonly used in the superficial and mucosal candidiasis treatment: mode of action and resistance development.

Recent progress in medical sciences and therapy resulted in an increased number of immunocompromised individuals. Candida albicans is the leading opportunistic fungal pathogen causing infections in humans, ranging from superficial mucosal lesions to disseminated or bloodstream candidiasis. Superficial candidiasis not always presents a risk to the life of the infected host, however it significantly lowers the quality of life. Superficial Candida infections are difficult to treat and their frequency of occurrence is currently rising. To implement successful treatment doctors should be up to date with better understanding of C. albicans resistance mechanisms. Despite high frequency of Candida infections there is a limited number of antimycotics available for therapy. This review focuses on current understanding of the mode of action and resistance mechanisms to conventional and emerging antifungal agents for treatment of superficial and mucosal candidiasis.

Cell wall carbohydrates content of pathogenic Candida albicans strain morphological forms.

INTRODUCTION: The study evaluated the cell wall carbohydrates fraction in blastoconidia grown in YEPD medium at 30 degrees C and in the conglomerate of true hyphae grown in human serum at 37 degrees C. MATERIAL AND METHODS: The clinical isolate obtained from a child with widespread C. albicans infection was used in the study. The cells were broken with glass beads, centrifuged to harvest the cell wall followed by subjection to TFA hydrolysis and in the result of that released monosaccharides were detected by HPAEC-PAD. Both, serum and temperature conditions (37 degrees C) affected germination process influencing the cell wall carbohydrates content when incubation in serum was prolonged from 1 to 18 h. RESULTS: The mannan content of blastoconidia was almost twofold higher compared to filamentous forms (149.25 +/- 299.24 vs 77.26 +/- 122.07). The glucan content was threefold lower in blastoconidia compared to hyphae (251.86 +/- 243.44 vs 755.81 +/- 1299.30). The chitin level was fourfold lower in blastoconidia compared to filaments (23.86 +/- 54.09 vs 106.29 +/- 170.12). The reason for the differences in the carbohydrates content may be related to type of morphology induced in different environmental conditions. Among tested carbohydrates, glucan appeared to be present in appreciably larger amounts in both tested morphological fractions. The ultrastructure of the blastoconidial cell wall revealed striking differences compared to the hyphae indicating the carbohydrates content alterations for wall assembly during hyphal growth at alkaline pH and temp. 37 degrees C. CONCLUSIONS: The study provided evidence for the relationship between morphogenesis, cell-cell adhesion induced by serum and changes in the level of carbohydrates content.

Structural damages in adsorbed vaccines affected by freezing.

This study was planned to evaluate structural damages in adsorbed vaccines affected by freezing using scanning electron microscopy and X-ray analysis of the elements. Randomly selected 42 vials of eight different types of WHO pre-qualified adsorbed freeze-sensitive vaccines from 10 manufacturers were included in the study. Vaccines were kept at 5 °C. Selected numbers of vials from each type were then exposed to -25 °C for 24 h periods. All samples were evaluated for their structure using scanning electron microscopy, X-ray analysis of the elements and precipitation time. Scanning electron microscopy of vaccines affected by freezing showed either smooth or rough surfaced conglomerates associated with phosphate content of the precipitate. These vaccines precipitated 2-15 times faster compared to non-frozen samples. Non-frozen samples showed uniform flocculent structure either dense or dispersed. X-ray analysis of precipitates in frozen samples confirmed that the precipitate is mainly aluminium clutters. Scanning electron microscopy confirmed that the lattice structure of bonds between adsorbent and the antigen is broken and aluminium forms conglomerates that grow in size and weight. The precipitation time of vaccines affected by freezing is 4.5 times faster on average compared to non-frozen samples. These facts form the basis of the "shake test".

Ultrastructure of Candida albicans pleomorphic forms: phase-contrast microscopy, scanning and transmission electron microscopy.

A modified method of glutaraldeyde-osmium tetroxide fixation was adjusted to characterize the ultrastructure of Candida albicans pleomorphic forms, using phase-contrast microscopy, scanning electron microscopy and transmission electron microscopy. The discovered morphological criteria defining the individual morphotypes are discussed in terms of mycological and histopathological diagnostics of candidiasis. The relations are discussed between fungal pleomorphism, virulence and susceptibility of different morphotypes to fungicides.

[Virulence factors of Candida albicans]. / Czynniki zjadliwosci Candida albicans.

Candida albicans is the most common etiological factor of opportunistic human fungal infections. In this review, we focus on the major virulence factors that mediate the pathogenesis of C. albicans. Among these virulence factors, secreted aspartyl proteases, adherence, pleomorphism are the most important features of C. albicans infections. Ability to exist as different pleomorphic forms is defined as pleomorphism. A number of quorum sensing (QS) molecules have been described which affect morphogenesis process in C. albicans. Furthermore, the morphological transition of C. albicans in response to changing environmental conditions represent a means by which the strain adapts to different biological niches. Furthermore, every morphotype has own virulence profile and each pleomorphic form provide critical functions required for pathogenesis. Candida albicans is a producer of extracellular hydrolytic enzymes. Among them lipases, phospholipases and secreted aspartyl proteinases (Sap) are most significant in virulence. Sap proteins contribute to pathogenesis by digestion of host cell membranes and molecules of the host immune system to avoid antimicrobial attack by the host. One of the key features in the development of candidiasis is adhesion ofC. albicans to buccal and vaginal epithelial cells. The adhesion to host cells represents the first step in the internalization process which involves adhesins. Knowledge of the role of the various C. albicans' virulence factors during in vivo infections is still incomplete, therefore further studies including quantification of genes expression and histopathological examination of tissues damage are required to fully understand pathogenesis of this opportunistic pathogen.

In vitro study of secreted aspartyl proteinases Sap1 to Sap3 and Sap4 to Sap6 expression in Candida albicans pleomorphic forms.

Transition from round budding cells to long hyphal forms and production of secreted aspartic proteases (Saps) are considered virulence-associated factors of Candida albicans. Although plenty of data dealing with Saps involvement in the infection process have been published, Saps expression by the different pleomorphic forms as well as the capacity of C. albicans filaments to express Sap1-6 under serum influence are poorly investigated. In this study, we used immunofluorescence and immunoelectron microscopy for the detection of Sap1-6 isoenzymes in C. albicans pleomorphic cells (blastoconidia, germ tubes, pseudohyphae, true hyphae) grown in Sap-inductive human serum and Sap non-inductive medium - yeast extract-peptone-glucose (YEPD). Isoenzymes were below the detection level in all blastoconidial cells grown in YEPD for 18 h. Sap1-6 expression was hardly detected in C. albicans cells cultivated in serum for 20 min. Increasing level of Sap1-6 expression was observed when C. albicans was incubated for 2, 6 and 18 h in serum corresponding to the development of germ tubes, pseudohyphae and true hyphae. The expression of Sap1-3 in pseudohyphae and true hyphae was more intensive compared to Sap4-6. Thus, we could show that human serum induced hyphae formation and the expression of Sap1-6 were co-regulated.

Validation of the shake test for detecting freeze damage to adsorbed vaccines.

OBJECTIVE: To determine the validity of the shake test for detecting freeze damage in aluminium-based, adsorbed, freeze-sensitive vaccines. METHODS: A double-blind crossover design was used to compare the performance of the shake test conducted by trained health-care workers (HCWs) with that of phase contrast microscopy as a "gold standard". A total of 475 vials of 8 different types of World Health Organization prequalified freeze-sensitive vaccines from 10 different manufacturers were used. Vaccines were kept at 5 degrees C. Selected numbers of vials from each type were then exposed to -25 degrees C and -2 degrees C for 24-hour periods. FINDINGS: There was complete concordance between HCWs and phase-contrast microscopy in identifying freeze-damaged vials and non-frozen samples. Non-frozen samples showed a fine-grain structure under phase contrast microscopy, but freeze-damaged samples showed large conglomerates of massed precipitates with amorphous, crystalline, solid and needle-like structures. Particles in the non-frozen samples measured from 1 microm (vaccines against diphtheria-tetanus-pertussis; Haemophilus influenzae type b; hepatitis B; diphtheria-tetanus-pertussis-hepatitis B) to 20 microm (diphtheria and tetanus vaccines, alone or in combination). By contrast, aggregates in the freeze-damaged samples measured up to 700 microm (diphtheria-tetanus-pertussis) and 350 microm on average. CONCLUSION: The shake test had 100% sensitivity, 100% specificity and 100% positive predictive value in this study, which confirms its validity for detecting freeze damage to aluminium-based freeze-sensitive vaccines.

[Prof. Wlodzimierz Kurylowicz (MD) (1910-1991)--100th anniversary of birthday]. / Prof. Dr hab. med. Wlodzimierz Kurylowicz (1910-1991)--na stulecie urodzin.

The death of Professor Wlodzimierz Kurylowicz on February 21, 1991 at the age of 80 was a great loss that is especially felt by all those who have been involved in the research of antibiotics. He is remembered as one of pioneers of the antibiotic era. Prof. W. Kurylowicz was born September 26, 1910 in Lvov. He received his MD from the Jan Kazimierz University in Lvov. Following posts as an Associate Professor of Microbiology at Lvov and as a research bacteriologist at the National Institute of Hygiene in Warsaw he became a Professor of Microbiology at that Institute. He was Director of the Institute from 1964 to 1980. More than 270 of this works regarded such topics as: antibiotic biogenesis and biosynthesis, numerical taxonomy of Streptomyces spp., evaluation of BCG and microbial fine structure. Prof. W. Kurylowicz was a recipient of the National Prize Award for scientific guidance in construction of the first antibiotic industry in Poland, and the National Prize Award for the co-authorship of the monograph "Antibiotics - Origin, Nature and Properties". He was also recipient of Doctorates honoris causa of: Nicolas Copernicus Medical University in Poland; University of Oslo; University of Lille; Medical University of Debrecen, Hungary; University of Liège, Belgium; Federal University of Pernambuco, Recife, Brazil; University of Quèbec, Canada; University of Münster, Germany.

Synthesis of 3-Substituted-clavams: Antifungal Properties, DD-Peptidase and beta-Lactamase Inhibition.

The [2+2]cycloaddition of chlorosulfonyl isocyanate to vinyl and (Z)-propenyl ethers derived from the 2-O-sulfonylated (R)- and (S)-1-(furyl-2')-1,2-ethanediols furnished the 4-alkoxy-azetidin-2-ones with a good to moderate stereoselectivity. The intramolecular alkylation of the beta-lactam nitrogen atom led to the corresponding 3-(furyl-2')- and 6-methyl-3-(furyl-2')-clavams. The transformation of the furyl residue into an alkoxycarbonyl group led to clavams related to the natural compounds. The synthesized clavams exhibited moderate inhibitory activities against DD-peptidase 64-575 and beta-lactamase (penase) as well as antifungal activities.

Protein production and secretion in an Aspergillus nidulans mutant impaired in glycosylation.

O-glycosylation has been considered a limiting factor in protein secretion in filamentous fungi. Overexpression of the yeast DPM1 gene encoding dolichylphosphate mannose synthase (DPMS) in an Aspergillus nidulans mutant (BWB26A) deficient in O-glycosylation caused an increase in the number of secretory vesicles and changes in protein secretion. However, the secretory proteins, primarily O-mannosylated glucoamylase and N-glycosylated invertase, were mainly trapped in the periplasmic space. Different glycoforms of invertase were found insite the cells, in the periplasmic space and in the cultivation medium. Our data point to the importance of the cell wall as a barrier in protein secretion.

Practical use of DD-peptidase 64-575 for the assay of inhibition activity of natural and synthetic beta-lactam compounds.

DD-peptidase 64-575 is produced by Saccharopolyspora erythrea PZH TZ 64-575. This enzyme is very sensitive to beta-lactam antibiotics. It could be used to assay the affinity of natural and synthetic beta-lactam compounds. In this paper an enzymatic method is described for investigation of synthetic, insoluble in water beta-lactams.