Surface Proteins of Lactobacillus crispatus: Adhesive Properties and Cell Wall Anchoring

Bacterial surface-associated proteins are important in communication with the environment and bacteria-host interactions. In this thesis work, surface molecules of Lactobacillus crispatus important in host interaction were studied. The L. crispatus strains of the study were known from previous studies to be efficient in adhesion to intestinal tract and ECM. L. crispatus JCM 5810 possess an adhesive surface layer (S-layer) protein, whose functions and domain structure was characterized. We cloned two S-layer protein genes (cbsA; collagen-binding S-layer protein A and silent cbsB) and identified the protein region in CbsA important for adhesion to host tissues, for polymerization into a periodic layer as well as for attachment to the bacterial cell surface. The analysis was done by extensive mutation analysis and by testing His6-tagged fusion proteins from recombinant Escherichia coli as well as by expressing truncated CbsA peptides on the surface of Lactobacillus casei. The N-terminal region (31-274) of CbsA showed efficient and specific binding to collagens, laminin and extracellular matrix on tissue sections of chicken intestine. The N-terminal region also contained the information for formation of periodic S-layer polymer. This region is bordered at both ends by a conserved short region rich in valines, whose substitution to leucines drastically affected the periodic polymer structure. The mutated CbsA proteins that failed to form a periodic polymer, did not bind collagens, which indicates that the polymerized structure of CbsA is needed for collagen-binding ability. The C-terminal region, which is highly identical in S-layer proteins of L. crispatus, Lactobacillus acidophilus and Lactobacillus helveticus, was shown to anchor the protein to the bacterial cell wall. The C-terminal CbsA peptide specifically bound to bacterial teichoic acid and lipoteichoic acids. In conclusion, the N-terminal domain of the S-layer protein of L. crispatus is important for polymerization and adhesion to host tissues, whereas the C-terminal domain anchors the protein to bacterial cell-wall teichoic acids. Lactobacilli are fermentative organisms that effectively lower the surrounding pH. While this study was in progress, plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified in the extracellular proteome of L. crispatus ST1. In this work, the cell-wall association of enolase and GAPDH were shown to rely on pH-reversible binding to the cell-wall lipoteichoic acids. Enolase from L. crispatus was functionally compared with enolase from L. johnsonii as well as from pathogenic streptococci (Streptococcus pneumoniae, Streptococcus pyogenes) and Staphylococcus aureus. His6-enolases from commensal lactobacilli bound human plasminogen and enhanced its activation by human plasminogen activators similarly to, or even better than, the enolases from pathogens. Similarly, the His6-enolases from lactobacilli exhibited adhesive characteristics previously assigned to pathogens. The results call for more detailed analyses of the role of the host plasminogen system in bacterial pathogenesis and commensalism as well of the biological role and potential health risk of the extracellular proteome in lactobacilli.

Compound heterozygous mutations in RIPPLY2 associated with vertebral segmentation defects

Segmentation defects of the vertebrae (SDV) are caused by aberrant somite formation during embryogenesis and result in irregular formation of the vertebrae and ribs. The Notch signal transduction pathway plays a critical role in somite formation and patterning in model vertebrates. In humans, mutations in several genes involved in the Notch pathway are associated with SDV, with both autosomal recessive (MESP2, DLL3, LFNG, HES7) and autosomal dominant (TBX6) inheritance. However, many individuals with SDV do not carry mutations in these genes. Using whole-exome capture and massive parallel sequencing, we identified compound heterozygous mutations in RIPPLY2 in two brothers with multiple regional SDV, with appropriate familial segregation. One novel mutation (c.A238T:p.Arg80*) introduces a premature stop codon. In transiently transfected C2C12 mouse myoblasts, the RIPPLY2 mutant protein demonstrated impaired transcriptional repression activity compared with wild-type RIPPLY2 despite similar levels of expression. The other mutation (c.240-4T>G), with minor allele frequency <0.002, lies in the highly conserved splice site consensus sequence 5' to the terminal exon. Ripply2 has a well-established role in somitogenesis and vertebral column formation, interacting at both gene and protein levels with SDV-associated Mesp2 and Tbx6. We conclude that compound heterozygous mutations in RIPPLY2 are associated with SDV, a new gene for this condition. © The Author 2014.

A Mycobacterial Cyclic AMP Phosphodiesterase That Moonlights as a Modifier of Cell Wall Permeability

Mycobacterium tuberculosis utilizes many mechanisms to establish itself within the macrophage, and bacterially derived cAMP is important in modulating the host cellular response. Although the genome of M. tuberculosis is endowed with a number of mammalian-like adenylyl cyclases, only a single cAMP phosphodiesterase has been identified that can decrease levels of cAMP produced by the bacterium. We present the crystal structure of the full-length and sole cAMP phosphodiesterase, Rv0805, found in M. tuberculosis, whose orthologs are present only in /the genomes of slow growing and pathogenic mycobacteria. The dimeric core catalytic domain of Rv0805 adopts a metallophosphoesterase fold, and the C-terminal region builds the active site and contributes to multiple substrate utilization.Localization of Rv0805 to the cell wall is dependent on its C terminus, and expression of either wild type or mutationally inactivated Rv0805 in M. smegmatis alters cell permeability to hydrophobic cytotoxic compounds. Rv0805 may therefore play a key role in the pathogenicity of mycobacteria, not only by hydrolyzing bacterial cAMP, but also by moonlighting as a protein that can alter cell wall functioning.

Reducing luminance contrast on the window wall and users’ interventions in an office room

High luminance contrast between windows and surrounding surfaces could cause discomfort glare, which could reduce office workers’ productivity. It might also increase energy usage of buildings due to occupants’ interventions in lighting conditions to improve indoor visual quality. It is presumed that increasing the luminance of the areas surrounding the windows using a supplementary system, such Light Emitting Diodes (LEDs), could reduce discomfort glare. This paper reports on the results of a pilot study in a conventional office in Brisbane, Australia. The outcomes of this study indicated that a supplementary LED system could reduce the luminance contrast on the window wall from values in the order of 24:1 to 12:1. The results suggest that this reduction could significantly reduce discomfort glare from windows, as well as diminishing the likelihood of users’ intention to turn on the ceiling lights and/ or to move the blind down.

Defects in the IFT-B component IFT172 cause Jeune and Mainzer-Saldino syndromes in humans

Intraflagellar transport (IFT) depends on two evolutionarily conserved modules, subcomplexes A (IFT-A) and B (IFT-B), to drive ciliary assembly and maintenance. All six IFT-A components and their motor protein, DYNC2H1, have been linked to human skeletal ciliopathies, including asphyxiating thoracic dystrophy (ATD; also known as Jeune syndrome), Sensenbrenner syndrome, and Mainzer-Saldino syndrome (MZSDS). Conversely, the 14 subunits in the IFT-B module, with the exception of IFT80, have unknown roles in human disease. To identify additional IFT-B components defective in ciliopathies, we independently performed different mutation analyses: candidate-based sequencing of all IFT-B-encoding genes in 1,467 individuals with a nephronophthisis-related ciliopathy or whole-exome resequencing in 63 individuals with ATD. We thereby detected biallelic mutations in the IFT-B-encoding gene IFT172 in 12 families. All affected individuals displayed abnormalities of the thorax and/or long bones, as well as renal, hepatic, or retinal involvement, consistent with the diagnosis of ATD or MZSDS. Additionally, cerebellar aplasia or hypoplasia characteristic of Joubert syndrome was present in 2 out of 12 families. Fibroblasts from affected individuals showed disturbed ciliary composition, suggesting alteration of ciliary transport and signaling. Knockdown of ift172 in zebrafish recapitulated the human phenotype and demonstrated a genetic interaction between ift172 and ift80. In summary, we have identified defects in IFT172 as a cause of complex ATD and MZSDS. Our findings link the group of skeletal ciliopathies to an additional IFT-B component, IFT172, similar to what has been shown for IFT-A.

Seismic behaviour of rigid-faced reinforced soil retaining wall models:reinforcement effect

This paper presents the results of shaking table tests on models of rigid-faced reinforced soil retaining walls in which reinforcement materials of different tensile strength were used. The construction of the model retaining walls in a laminar box mounted on a shaking table, the instrumentation and the results from the shaking table tests are described in detail and the effects of the reinforcement parameters on the acceleration response at different elevations of the retaining wall, horizontal soil pressures and face deformations are presented. It was observed from these tests that the horizontal face displacement response of the rigid-faced retaining walls was significantly affected by the inclusion of reinforcement and even low-strength polymer reinforcement was found to be efficient in significantly reducing the deformation of the face. The acceleration amplifications were, however, observed to be less influenced by the reinforcement parameters. The results obtained from this study are helpful in understanding the relative performance of reinforced soil retaining walls under the different test conditions used in the experiments.

Field Emission Properties of Carbon Nanotube Arrays with Defects and Impurities

It has been found experimentally that the results related to the collective field emission performance of carbon nanotube (CNT) arrays show variability. The emission performance depends on the electronic structure of CNTs (especially their tips). Due to limitations in the synthesis process, production of highly pure and defect free CNTs is very difficult. The presence of defects and impurities affects the electronic structure of CNTs. Therefore, it is essential to analyze the effect of defects on the electronic structure, and hence, the field emission current. In this paper, we develop a modeling approach for evaluating the effect of defects and impurities on the overall field emission performance of a CNT array. We employ a concept of effective stiffness degradation for segments of CNTs, which is due to structural defects. Then, we incorporate the vacancy defects and charge impurity effects in our Green's function based approach. Simulation results indicate decrease in average current due to the presence of such defects and impurities.

Postoperative soft tissue defects in the ankle area : The etiology and methods of reconstruction

The risk is obvious for soft tissue complications after operative treatment of the Achilles tendon, calcaneal bone or after ankle arthroplasty. Such complications after malleolar fractures are, however, seldom seen. The reason behind these complications is that the soft tissue in this region is tight and does not allow much tension to the wound area after surgery. Furthermore the area of operation may be damaged by swelling after the injury, or can be affected by peripheral vascular disease. While complications in this area are unavoidable, they can be diminished. This study attempts to highlight the possible predisposing factors leading to complications in these operations and on the other hand, to determine the solutions to solve soft tissue problems in this region. The study consists of five papers. The first article is a reprint on the soft tissue reconstruction of 25 patients after their complicated Achilles tendon surgeries were analysed. The second study reviews a series of 126 patients after having undergone an operative treatment of calcaneal bone fractures and analyses the complications and possible reasons behind them. The third part analyses a series of corrections of 35 soft tissue complications after calcaneal fracture operations. The fourth part reviews a series of 7 patients who had undergone complicated ankle arthroplasties. The last article presents a series of post operative lateral defects of the ankle treated with a less frequently used distally based peroneus brevis muscle flap and analyses the results. What can be conducted from these studies is that in general, the results after the correction of even severe soft tissue complications in the ankle region are good. For the small defects around the Achilles tendon, the local flaps are useful, but the larger defects are best treated with a free flap. We found that a long delay from trauma to surgery and a long operating time were predisposing factors that lead to soft tissue complications after operatively treated calcaneal bone fractures. The more severe the injury, the greater the risk for wound complication. Surprisingly, the long-term results after infected calcaneal osteosyntheses were acceptable and the calcaneal bone seems to tolerate chronic infections very well if the soft tissue is reconstructed successfully. Behind the complicated ankle arthroplasties, unexpectedly high number of cases experiencing arteriosclerosis of the lower extremity was found. These complications lead to ankle fusion but can be solved with a free flap if the vascularity is intact or can be reconstructed. For this reason a vascular examination of the lower extremity arteries of the patients going to ankle arthroplasty is strongly recommended. Moreover postoperative lateral malleolar wound infections which typically create lateral ankle defects can successfully be treated with a peroneus brevis muscle flap covered with a free skin graft.

Outcomes of ruptured abdominal aortic aneurysm

Ruptured abdominal aortic aneurysm (RAAA) is a life-threatening event, and without operative treatment the patient will die. The overall mortality can be as high as 80-90%; thus repair of RAAA should be attempted whenever feasible. The quality of life (QoL) has become an increasingly important outcome measure in vascular surgery. Aim of the study was to evaluate outcomes of RAAA and to find out predictors of mortality. In Helsinki and Uusimaa district 626 patients were identified to have RAAA in 1996-2004. Altogether 352 of them were admitted to Helsinki University Central Hospital (HUCH). Based on Finnvasc Registry, 836 RAAA patients underwent repair of RAAA in 1991-1999. The 30-day operative mortality, hospital and population-based mortality were assessed, and the effect of regional centralisation and improving in-hospital quality on the outcome of RAAA. QoL was evaluated by a RAND-36 questionnaire of survivors of RAAA. Quality-adjusted life years (QALYs), which measure length and QoL, were calculated using the EQ-5D index and estimation of life expectancy. The predictors of outcome after RAAA were assessed at admission and 48 hours after repair of RAAA. The 30-day operative mortality rate was 38% in HUCH and 44% nationwide, whereas the hospital mortality was 45% in HUCH. Population-based mortality was 69% in 1996-2004 and 56% in 2003-2004. After organisational changes were undertaken, the mortality decreased significantly at all levels. Among the survivors, the QoL was almost equal when compared with norms of age- and sex-matched controls; only physical functioning was slightly impaired. Successful repair of RAAA gave a mean of 4.1 (0-30.9) QALYs for all RAAA patients, although non-survivors were included. The preoperative Glasgow Aneurysm Score was an independent predictor of 30-day operative mortality after RAAA, and it also predicted the outcome at 48- hours for initial survivors of repair of RAAA. A high Glasgow Aneurysm Score and high age were associated with low numbers of QALYs to be achieved. Organ dysfunction measured by the Sequential Organ Failure Assessment (SOFA) score at 48 hours after repair of RAAA was the strongest predictor of death. In conclusion surgery of RAAA is a life-saving and cost-effective procedure. The centralisation of vascular emergencies improved the outcome of RAAA patients. The survivors had a good QoL after RAAA. Predictive models can be used on individual level only to provide supplementary information for clinical decision-making due to their moderate discriminatory value. These results support an active operation policy, as there is no reliable measure to predict the outcome after RAAA.

Inflammatory and coagulation disturbances in acute pancreatitis

Acute pancreatitis (AP), a common cause of acute abdominal pain, is usually a mild, self-limited disease. However, some 20-30% of patients develop a severe disease manifested by pancreatic necrosis, abscesses or pseudocysts, and/or extrapancreatic complications, such as vital organ failure (OF). Patients with AP develop systemic inflammation, which is considered to play a role in the pathogenesis of multiple organ failure (MOF). OF mimics the condition seen in patients with sepsis, which is characterized by an overwhelming production of inflammatory mediators, activation of the complement system and systemic activation of coagulation, as well as the development of disseminated intravascular coagulation (DIC) syndrome. Vital OF is the major cause of mortality in AP, along with infectious complications. About half of the deaths occur within the first week of hospitalization and thus, early identification of patients likely to develop OF is important. The aim of the present study was to investigate inflammatory and coagulation disturbances in AP and to find inflammatory and coagulation markers for predicting severe AP, and development of OF and fatal outcome. This clinical study consists of four parts. All of patients studied had AP when admitted to Helsinki University Central Hospital. In the first study, 31 patients with severe AP were investigated. Their plasma levels of protein C (PC) and activated protein C (APC), and monocyte HLA-DR expression were studied during the treatment period in the intensive care unit; 13 of these patients developed OF. In the second study, the serum levels of complement regulator protein CD59 were studied in 39 patients during the first week of hospitalization; 12 of them developed OF. In the third study, 165 patients were investigated; their plasma levels of soluble form of the receptor for advanced glycation end products (sRAGE) and high mobility group box 1 (HMGB1) protein were studied during the first 12 days of hos-pitalization; 38 developed OF. In the fourth study, 33 patients were studied on admission to hospital for plasma levels of prothrombin fragment F1+2 and tissue factor pathway inhibitor (TFPI), and thrombin formation capacity by calibrated automated thrombogram (CAT); 9 of them developed OF. Our results showed significant PC deficiency and decreased APC generation in patients with severe AP. The PC pathway defects seemed to be associated with the development of OF. In patients who developed OF, the levels of serum CD59 and plasma sRAGE, but not of HMGB1, were significantly higher than in patients who recovered without OF. The high CD59 levels on admission to the hospital seemed to be predictive for severe AP and OF. The median of the highest sRAGE levels was significantly higher in non-survivors than in survivors. No significant difference between the patient groups was found in the F1+2 levels. The thrombograms of all patients were disturbed in their shape, and in 11 patients the exogenous tissue factor did not trigger thrombin generation at all ( flat curve ). All of the patients that died displayed a flat curve. Free TFPI levels and free/total TFPI ratios were significantly higher in patients with a flat curve than in the others, and these levels were also significantly higher in non-survivors than in survivors. The flat curve in combination with free TFPI seemed to be predictive for a fatal outcome in AP.

Time Domain Characteristics of Electrical Measures for a Piezoelectric Thin Film to Identify Defects in the Substrate

A reduced 3D continuum model of dynamic piezoelectricity in a thin-film surface-bonded to the substrate/host is presented in this article. While employing large area flexible thin piezoelectric films for novel applications in device/diagnostics, the feasibility of the proposed model in sensing the surface and/or sub-surface defects is demonstrated through simulations - which involve metallic beams with cracks and composite beam with delaminations of various sizes. We have introduced a set of electrical measures to capture the severity of the damage in the existing structures. Characteristics of these electrical measures in terms of the potential difference and its spatial gradients are illustrated in the time domain. Sensitivity studies of the proposed measures in terms of the defected areas and their region of occurence relative to the sensing film are reported. The simulations' results for electrical measures for damaged hosts/substrates are compared with those due to undamaged hosts/substrates, which show monotonicity with high degree of sensitivity to variations in the damage parameters.