Biotransformation of protein-rich waste by Yarrowia lipolytica IPS21 to high-value products-amino acid supernatants.

The yeast strain Yarrowia lipolytica IPS 21 was tested for its ability to degrade potentially toxic chrome-tanned leather shavings (CTLS) in a liquid environment. Biological and chemical parameters were monitored during a 48-h period of biotransformation of the protein-rich waste. CTLS was added at a concentration of 0.1-4% (wt/wt) to a modified YPG medium (15 g L-1 yeast extract and 5 g L-1 NaCl). Biodegradation and bioconversion were performed in a one-step process. It was found that the higher degradation rate depended on the activity of the proteases and the pH of the medium, but not on the initial inoculum ratio and the activity of the dehydrogenase. The highest efficiency of the process was obtained for 4% (wt/wt) CTLS on day 2 (degradation rate 58-67%, biomass production 2.11-2.20 g L-1, protease activity 312 U mg-1 protein, and pH 9.20). Our results showed that total chromium was probably not transported across the cytoplasmic membrane of Y. lipolytica IPS21 and that chromium (III) was not oxidized to chromium (VI). The phytotoxicity of selected amino acid supernatants [2.5% (vol/vol)] was tested after the bioconversion process. It was found that the supernatants had a stimulating effect on the plants tested. The root elongation was 29-28% higher than that of the reference samples. This result makes Y. lipolytica IPS21 a potential candidate for safely converting potentially toxic protein-rich wastes into valuable products without enzyme isolation, e.g., amino acid fertilizers. IMPORTANCE Enzyme technologies have the greatest practical relevance to environmental trends. Overcoming the barrier of the high cost of carbon substrates used for biotransformation is the main challenge of these methods. The huge potential of the use of extracellular proteases of Yarrowia species or amino acids in various industries indicates the need for the extension of basic research on waste as a carbon source for this yeast. The experiments demonstrated that it is possible to use Y. lipolytica IPS21 for bioconversion of chrome-tanned leather shavings (CTLS) in a single-step process and to produce high-value amino acid supernatant without having an isolated enzyme. In our study, we show the effect of 2.5% (vol/vol) CTLS supernatant obtained from Y. lipolytica IPS21 on the elongation of the root system of selected plants and provide information on the effect of environmental factors on the efficiency of the bioconversion and the migration of chromium.

Exposure to stressors and antimicrobials induces cell-autonomous ultrastructural heterogeneity of an intracellular bacterial pathogen.

Despite their clonality, intracellular bacterial pathogens commonly show remarkable physiological heterogeneity during infection of host cells. Physiological heterogeneity results in distinct ultrastructural morphotypes, but the correlation between bacterial physiological state and ultrastructural appearance remains to be established. In this study, we showed that individual cells of Salmonella enterica serovar Typhimurium are heterogeneous in their ultrastructure. Two morphotypes based on the criterion of cytoplasmic density were discriminated after growth under standard culture conditions, as well as during intracellular lifestyle in mammalian host cells. We identified environmental conditions which affect cytoplasmic densities. Using compounds generating oxygen radicals and defined mutant strains, we were able to link the occurrence of an electron-dense ultrastructural morphotype to exposure to oxidative stress and other stressors. Furthermore, by combining ultrastructural analyses of Salmonella during infection and fluorescence reporter analyses for cell viability, we provided evidence that two characterized ultrastructural morphotypes with electron-lucent or electron-dense cytoplasm represent viable cells. Moreover, the presence of electron-dense types is stress related and can be experimentally induced only when amino acids are available in the medium. Our study proposes ultrastructural morphotypes as marker for physiological states of individual intracellular pathogens providing a new marker for single cell analyses.

ATRT-SHH comprises three molecular subgroups with characteristic clinical and histopathological features and prognostic significance.

Atypical teratoid/rhabdoid tumor (ATRT) is an aggressive central nervous system tumor characterized by loss of SMARCB1/INI1 protein expression and comprises three distinct molecular groups, ATRT-TYR, ATRT-MYC and ATRT-SHH. ATRT-SHH represents the largest molecular group and is heterogeneous with regard to age, tumor location and epigenetic profile. We, therefore, aimed to investigate if heterogeneity within ATRT-SHH might also have biological and clinical importance. Consensus clustering of DNA methylation profiles and confirmatory t-SNE analysis of 65 ATRT-SHH yielded three robust molecular subgroups, i.e., SHH-1A, SHH-1B and SHH-2. These subgroups differed by median age of onset (SHH-1A: 18 months, SHH-1B: 107 months, SHH-2: 13 months) and tumor location (SHH-1A: 88% supratentorial; SHH-1B: 85% supratentorial; SHH-2: 93% infratentorial, often extending to the pineal region). Subgroups showed comparable SMARCB1 mutational profiles, but pathogenic/likely pathogenic SMARCB1 germline variants were over-represented in SHH-2 (63%) as compared to SHH-1A (20%) and SHH-1B (0%). Protein expression of proneural marker ASCL1 (enriched in SHH-1B) and glial markers OLIG2 and GFAP (absent in SHH-2) as well as global mRNA expression patterns differed, but all subgroups were characterized by overexpression of SHH as well as Notch pathway members. In a Drosophila model, knockdown of Snr1 (the fly homologue of SMARCB1) in hedgehog activated cells not only altered hedgehog signaling, but also caused aberrant Notch signaling and formation of tumor-like structures. Finally, on survival analysis, molecular subgroup and age of onset (but not ASCL1 staining status) were independently associated with overall survival, older patients (> 3 years) harboring SHH-1B experiencing relatively favorable outcome. In conclusion, ATRT-SHH comprises three subgroups characterized by SHH and Notch pathway activation, but divergent molecular and clinical features. Our data suggest that molecular subgrouping of ATRT-SHH has prognostic relevance and might aid to stratify patients within future clinical trials.

A versatile remote control system for functional expression of bacterial virulence genes based on the tetA promoter.

The expression of bacterial virulence factors is controlled in response to host or environmental factors and most virulence genes are not expressed under laboratory conditions. Investigations of molecular structures and cellular functions of bacterial virulence factors demand systems for experimentally controlled expression. We describe a simple and robust system that is based on the tetA promoter and the cognate repressor TetR. Expression under control of PtetA can be induced by non-antibiotic derivatives of tetracycline such as anhydrotetracycline (AHT). Tet-on expression cassettes can be used to replace native promoters of chromosomal genes or operons of interest. Tet-on plasmids allow episomal expression in homologous or heterologous host organisms. We demonstrate the application of Tet-on systems for the controlled induction of flagella assembly and motility, and for surface expression of adhesins of the chaperone/usher family of enteropathogenic Escherichia coli and autotransporter adhesins of Yersinia enterocolitica in Salmonella enterica and E. coli. Since inducer AHT can easily cross bacterial envelopes and mammalian cell membranes, the system can also be applied to control virulence genes in intracellular bacteria. We demonstrate the controlled synthesis, translocation and function of effector proteins of the type III secretion system of intracellular S. enterica.

ER Lipid Defects in Neuropeptidergic Neurons Impair Sleep Patterns in Parkinson's Disease.

Parkinson's disease patients report disturbed sleep patterns long before motor dysfunction. Here, in parkin and pink1 models, we identify circadian rhythm and sleep pattern defects and map these to specific neuropeptidergic neurons in fly models and in hypothalamic neurons differentiated from patient induced pluripotent stem cells (iPSCs). Parkin and Pink1 control the clearance of mitochondria by protein ubiquitination. Although we do not observe major defects in mitochondria of mutant neuropeptidergic neurons, we do find an excess of endoplasmic reticulum-mitochondrial contacts. These excessive contact sites cause abnormal lipid trafficking that depletes phosphatidylserine from the endoplasmic reticulum (ER) and disrupts the production of neuropeptide-containing vesicles. Feeding mutant animals phosphatidylserine rescues neuropeptidergic vesicle production and acutely restores normal sleep patterns in mutant animals. Hence, sleep patterns and circadian disturbances in Parkinson's disease models are explained by excessive ER-mitochondrial contacts, and blocking their formation or increasing phosphatidylserine levels rescues the defects in vivo.

Functional expression of the entire adhesiome of Salmonella enterica serotype Typhimurium.

Adhesins are crucial virulence factors of pathogenic bacteria involved in colonization, transmission and pathogenesis. Many bacterial genomes contain the information for a surprisingly large number of diverse adhesive structures. One prominent example is the invasive and facultative intracellular pathogen Salmonella enterica with an adhesiome of up to 20 adhesins. Such large repertoire of adhesins contributes to colonization of a broad range of host species and may allow adaptation to various environments within the host, as well as in non-host environments. For S. enterica, only few members of the adhesiome are functionally expressed under laboratory conditions, and accordingly the structural and functional understanding of the majority of adhesins is sparse. We have devised a simple and versatile approach to functionally express all adhesins of S. enterica serotype Typhimurium, either within Salmonella or within heterologous hosts such as Escherichia coli. We demonstrate the surface expression of various so far cryptic adhesins and show ultrastructural features using atomic force microscopy and transmission electron microscopy. In summary, we report for the first time the expression of the entire adhesiome of S. enterica serotype Typhimurium.

Tau association with synaptic vesicles causes presynaptic dysfunction.

Tau is implicated in more than 20 neurodegenerative diseases, including Alzheimer's disease. Under pathological conditions, Tau dissociates from axonal microtubules and missorts to pre- and postsynaptic terminals. Patients suffer from early synaptic dysfunction prior to Tau aggregate formation, but the underlying mechanism is unclear. Here we show that pathogenic Tau binds to synaptic vesicles via its N-terminal domain and interferes with presynaptic functions, including synaptic vesicle mobility and release rate, lowering neurotransmission in fly and rat neurons. Pathological Tau mutants lacking the vesicle binding domain still localize to the presynaptic compartment but do not impair synaptic function in fly neurons. Moreover, an exogenously applied membrane-permeable peptide that competes for Tau-vesicle binding suppresses Tau-induced synaptic toxicity in rat neurons. Our work uncovers a presynaptic role of Tau that may be part of the early pathology in various Tauopathies and could be exploited therapeutically.

Mumps, measles and rubella vaccination in children with PFAPA syndrome.

There is no published data regarding immunologic response to vaccinations in children with PFAPA syndrome (periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis). The aim of this study was to evaluate mumps, measles and rubella immunity in children with PFAPA. 31 children with PFAPA syndrome and 22 healthy children (control group - CG) were recruited to the study. All children were previously vaccinated with one dose of MMR vaccine according to the Polish obligatory vaccination schedule. The patients from both groups were evaluated for anti-measles, anti-mumps and anti-rubella IgG antibodies concentrations (ELISA tests; the reference values for protective antibody levels were 150IU/L, 16RU/L and 11IU/ml respectively). The percentage of patients with protective antibodies levels was as follows: measles - 93.55% of PFAPA and 95.45% of CG patients (p=0.77); mumps - 74.19% of PFAPA and 95.45% of CG patients (p=0.02); rubella - 80.65% of PFAPA and 90.9% of CG patients (p=0.30). CONCLUSIONS: Children with PFAPA syndrome present a good response to the measles and rubella component of the MMR vaccine, however immunity against mumps after one dose of MMR may not be sufficient. Further investigation concerning immunity against vaccine-preventable diseases and the safety of vaccinations in children with periodic fever syndromes is required.

Hsc70-4 Deforms Membranes to Promote Synaptic Protein Turnover by Endosomal Microautophagy.

Synapses are often far from their cell bodies and must largely independently cope with dysfunctional proteins resulting from synaptic activity and stress. To identify membrane-associated machines that can engulf synaptic targets destined for degradation, we performed a large-scale in vitro liposome-based screen followed by functional studies. We identified a presynaptically enriched chaperone Hsc70-4 that bends membranes based on its ability to oligomerize. This activity promotes endosomal microautophagy and the turnover of specific synaptic proteins. Loss of microautophagy slows down neurotransmission while gain of microautophagy increases neurotransmission. Interestingly, Sgt, a cochaperone of Hsc70-4, is able to switch the activity of Hsc70-4 from synaptic endosomal microautophagy toward chaperone activity. Hence, Hsc70-4 controls rejuvenation of the synaptic protein pool in a dual way: either by refolding proteins together with Sgt, or by targeting them for degradation by facilitating endosomal microautophagy based on its membrane deforming activity.

A review of guidance on immunization in persons with defective or deficient splenic function.

The spleen acts as a blood filter and lymphopoietic organ. Asplenic and hyposplenic individuals are more susceptible to serious infections caused by encapsulated bacteria but they can be protected by antibiotic prophylaxis and immunizations. Recent progress in vaccinology means prophylaxis is now successful in the vast majority of serious infections with pneumococci, meningococci and Haemophilus influenzae type b responsible for the majority of cases of overwhelming sepsis in asplenic patients. Current guidelines are coherent. Physicians treating patients with conditions associated with hyposplenism are ethically obliged to immunize their patients using the vaccines currently available to protect them from largely preventable, life-threatening infections.

Synaptic Contacts Enhance Cell-to-Cell Tau Pathology Propagation.

Accumulation of insoluble Tau protein aggregates and stereotypical propagation of Tau pathology through the brain are common hallmarks of tauopathies, including Alzheimer's disease (AD). Propagation of Tau pathology appears to occur along connected neurons, but whether synaptic contacts between neurons are facilitating propagation has not been demonstrated. Using quantitative in vitro models, we demonstrate that, in parallel to non-synaptic mechanisms, synapses, but not merely the close distance between the cells, enhance the propagation of Tau pathology between acceptor hippocampal neurons and Tau donor cells. Similarly, in an artificial neuronal network using microfluidic devices, synapses and synaptic activity are promoting neuronal Tau pathology propagation in parallel to the non-synaptic mechanisms. Our work indicates that the physical presence of synaptic contacts between neurons facilitate Tau pathology propagation. These findings can have implications for synaptic repair therapies, which may turn out to have adverse effects by promoting propagation of Tau pathology.

Safety of oral ibuprofen--analysis of data from the spontaneous reporting system in Poland.

Ibuprofen is a popular over-the-counter, non-steroidal anti-inflammatory medication, frequently used for the relief of fever, headaches, menstrual and other minor pains as well as a major active ingredient in numerous cold preparations. We analyzed sales volume and data obtained from the monitoring of spontaneous reports on the adverse effects of IBUM soft capsules, IBUM Forte soft capsules, and IBUM oral suspension 100 mg/5 mL collected by the manufacturer (PPF HASCO-LEK S.A. Wroclaw, Poland) and National Monitoring Center in Warszawa in the period between October 2002 and June 2012. A total of 19,644,797 units of IBUM soft capsules 200 mg, 5,678,164 units of IBUM Forte soft capsules 400 mg and 4,333,325 units of IBUM oral suspension 100 mg/5 mL (29,656,286 units altogether) produced by PPF HASCO-LEK S.A. Wrodcaw, P'oland were marketed during the period analyzed. There were 5 spontaneous reports regarding these medications registered in Poland in the period analyzed. Forms of oral ibuprofen are very safe medication rarely causing adverse effects; nevertheless, the existing spontaneous monitoring system of adverse effects in Poland is not sensitive enough to detect all adverse effects and needs improvement.

HDAC6 is a Bruchpilot deacetylase that facilitates neurotransmitter release.

Presynaptic densities are specialized structures involved in synaptic vesicle tethering and neurotransmission; however, the mechanisms regulating their function remain understudied. In Drosophila, Bruchpilot is a major constituent of the presynaptic density that tethers vesicles. Here, we show that HDAC6 is necessary and sufficient for deacetylation of Bruchpilot. HDAC6 expression is also controlled by TDP-43, an RNA-binding protein deregulated in amyotrophic lateral sclerosis (ALS). Animals expressing TDP-43 harboring pathogenic mutations show increased HDAC6 expression, decreased Bruchpilot acetylation, larger vesicle-tethering sites, and increased neurotransmission, defects similar to those seen upon expression of HDAC6 and opposite to hdac6 null mutants. Consequently, reduced levels of HDAC6 or increased levels of ELP3, a Bruchpilot acetyltransferase, rescue the presynaptic density defects in TDP-43-expressing flies as well as the decreased adult locomotion. Our work identifies HDAC6 as a Bruchpilot deacetylase and indicates that regulating acetylation of a presynaptic release-site protein is critical for maintaining normal neurotransmission.

Morphometry of human musculus gluteus maximus in foetal period.

BACKGROUND: Magnus gluteal muscle (musculus gluteus maximus) belongs to the group of lower limb girdle muscles. It is one of the biggest muscles in human organism and is located mostly superficially in gluteal region. Literature provides discussion concerning its role in movement such as walking, running and climbing as well as plastic surgery in reconstructive operations of trochanter. Magnus gluteal muscle plays an important role in orthopaedic surgery. OBJECTIVES: The goal of the study was to analyse the human magnus gluteal muscle in the foetal period. MATERIAL AND METHODS: The analysis was carried out on 154 muscles originating from human foetuses (including 30 females - 39%) belonging to the collection of Normal Anatomy Dept. of Wroclaw Medical University. The body length was assessed with the use of vertex-tuberal (v-tub) length and it was included in the range 107-205 mm, which corresponds with the period 17-30 weeks of foetal life. The survey incorporated the following methods: anthropological, preparational and image acquisition which was acquired with the use of high-resolution digital camera. In order to take computer measurements, the following systems were exploited: Image J and Scion for Windows. Statistical analysis was carried out with the use of STATISTICA package v. 9 (t-Student test). RESULTS: The magnus gluteal muscle was analysed in respect to sexual dimorphism and symmetry. On the basis of elicited parameters, the model of muscle increase in foetal period was defined. The following measurements were taken: v-tub, vertex-plantare (v-pl), body mass, muscle particular sides lengths and distance between corresponding measurement points. In every muscle, the lengths of four sections forming the circumference as well as the area were measured. CONCLUSIONS: No difference was observed in foetal magnus gluteal muscle sexual dimorphism or symmetry (p > 0.05). The correlation diagram was used to calculate the muscle weekly increase in foetal period. The results suggest that lesions and pathologies in the region of magnus gluteal muscle are acquired in post foetal period.

Dynamin photoinactivation blocks Clathrin and α-adaptin recruitment and induces bulk membrane retrieval.

Dynamin is a well-known regulator of synaptic endocytosis. Temperature-sensitive dynamin (shi(ts1)) mutations in Drosophila melanogaster or deletion of some of the mammalian Dynamins causes the accumulation of invaginated endocytic pits at synapses, sometimes also on bulk endosomes, indicating impaired membrane scission. However, complete loss of dynamin function has not been studied in neurons in vivo, and whether Dynamin acts in different aspects of synaptic vesicle formation remains enigmatic. We used acute photoinactivation and found that loss of Dynamin function blocked membrane recycling and caused the buildup of huge membrane-connected cisternae, in contrast to the invaginated pits that accumulate in shi(ts1) mutants. Moreover, photoinactivation of Dynamin in shi(ts1) animals converted these pits into bulk cisternae. Bulk membrane retrieval has also been seen upon Clathrin photoinactivation, and superresolution imaging indicated that acute Dynamin photoinactivation blocked Clathrin and α-adaptin relocalization to synaptic membranes upon nerve stimulation. Hence, our data indicate that Dynamin is critically involved in the stabilization of Clathrin- and AP2-dependent endocytic pits.

Respiratory complications in children hospitalized with varicella.

The aim of the study was to retrospectively determine the incidence and clinical course of varicella-related respiratory complications in children during the 6-year period 2005-2010. We attempted to identify the predisposing factors and outcome of such complications. Clinical records of 237 children treated in an academic hospital of the Medical University in Wroclaw, Poland were reviewed, taking into consideration the reason for referral to the hospital, duration of hospitalization, and diagnosis. There were 28 (11.8 %) children (mean age 2.8 ± 2.8 years) in the cohort hospitalized with varicella-related respiratory complications. The infants younger than 1 year predominated (9/28). None of the children were previously immunized against varicella. Admission occurred 5.0 ± 2.8 days after the first symptoms of varicella. The source of infection was an older sibling in 13/28 cases. The mean duration of hospitalization was 5.4 ± 2.0 days. The main symptoms were fever (20/28), cough (26/28), tachypnea (11/28), and dyspnea (7/28). Chest X-ray was performed in eight children, confirming pneumonia in six cases. Based on blood gases, chest X-ray, and clinical symptoms, pneumonia was diagnosed in 15/28 and acute bronchitis in 8/28 children. Intravenous antiviral therapy with acyclovir was administered in 16/28 and antibiotics in 14/28 children. In two cases, oxygen therapy was required and one child presented respiratory failure treated in the Intensive Care Unit. We conclude that respiratory tract involvement in the course of varicella infection in children is relatively common. Age less than 1 and an infected older sibling seem major risk factors for respiratory complications.

Immunopathology of exercise-induced bronchoconstriction in athletes--a new modified inflammatory hypothesis.

Elite athletes have a higher prevalence of exercise-induced bronchoconstriction than the general population. The pathogenesis of exercise-induced bronchoconstriction is not fully elucidated. Increasing evidence suggests that airway inflammation plays a major role in the immunopathogenesis of exercise-induced bronchoconstriction. The aim of our review is to discuss existing evidence and to present a new, modified inflammatory hypothesis of exercise-induced bronchoconstriction. Exercise alters the number and function of circulating immune cells. Episodes of upper respiratory symptoms in elite athletes do not follow the usual seasonal patterns. Moreover, they have an unusual short-term duration, which suggests a non-infectious etiology. If the pro-inflammatory response to exercise has the potential to induce symptoms that mimic respiratory tract infection, it definitely up-regulates pro-inflammatory cytokine expression in the airways. We can conclude that exercise up-regulates airway cytokine expression in a way that favors inflammation and allergic reactions in bronchi and lowers the threshold for bronchoconstriction to different stimuli like cool, dry air, allergens, and pollutants.

Influenza immunization rates in children and teenagers in Polish cities: conclusions from the 2009/2010 season.

The aim of this study was to determine influenza vaccine coverage among children aged 0-18 years in inner city practices in Poland in the 2009/2010 season and factors that might have influenced low vaccination coverage. A retrospective review of 11,735 vaccination charts of children aged 0-18 from seven randomly selected general practices in the capital city of Warsaw and one large practice in the city of Wroclaw was performed. We calculated the numbers of children who were vaccinated in the 2009/2010 season and analyzed the age distribution of vaccinated children. We also reviewed the vaccination history in patients who were vaccinated against influenza including: previous influenza vaccinations, modification (widening) of standard immunization scheme, and a proportion of children who completed the recommended two-dose schedule of vaccination. In the calculations, 95% confidence intervals were used. Out of the total of 11,735 children surveyed, 362 (3.1%, CI: 2.8-3.4%) were vaccinated against influenza in the 2009/2010 season. For 115 of these 362 (31.8%, CI: 27.0-36.6%) children it was their first vaccination against influenza. The mean age of a vaccinated child was 6.0 ± 4.3 years. Children aged 2-5 were most commonly vaccinated (153/362, 42.3%, CI: 37.2-47.4%), while infants (aged 6-12 months) were vaccinated rarely (15/362, 4.4%, CI: 2.2-6.2%). In the group of children younger than 8 years (86/362 children) who were vaccinated for the first time in their life only 29/86 (33.7%, CI: 23.7-43.7%) completed the recommended two-dose schedule. In conclusion, the importance of vaccinating children against influenza is hugely understated in Poland. General physicians should actively recommend annual influenza immunization of children. Recommendations of National Immunization Program concerning influenza vaccine should be clearer, simpler, and easier to implement.

LRRK2 controls an EndoA phosphorylation cycle in synaptic endocytosis.

LRRK2 is a kinase mutated in Parkinson's disease, but how the protein affects synaptic function remains enigmatic. We identified LRRK2 as a critical regulator of EndophilinA. Using genetic and biochemical studies involving Lrrk loss-of-function mutants and Parkinson-related LRRK2(G2019S) gain-of-kinase function, we show that LRRK2 affects synaptic endocytosis by phosphorylating EndoA at S75, a residue in the BAR domain. We show that LRRK2-mediated EndoA phosphorylation has profound effects on EndoA-dependent membrane tubulation and membrane association in vitro and in vivo and on synaptic vesicle endocytosis at Drosophila neuromuscular junctions in vivo. Our work uncovers a regulatory mechanism that indicates that reduced LRRK2 kinase activity facilitates EndoA membrane association, while increased kinase activity inhibits membrane association. Consequently, both too much and too little LRRK2-dependent EndoA phosphorylation impedes synaptic endocytosis, and we propose a model in which LRRK2 kinase activity is part of an EndoA phosphorylation cycle that facilitates efficient vesicle formation at synapses.