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All living cells depend on the intricate organization of molecular components in space and time. Although this notion was historically based on eukaryotic cells, with their structured intracellular architecture and cellular morphologies, it is now recognized that prokaryotes (that is, bacteria and archaea) also possess complex structures. A cytoskeleton is a network of intracellular protein filaments that play a structural or mechanical role (such as scaffolding, pushing, or pulling) in the spatiotemporal organization of cellular processes. Polymerization of protein monomers in a roughly linear fashion into filaments represents an effective means to establish long-range spatial order by bridging the gap between nanometer-sized molecules and micron-sized cells. It is now evident that bacteria and archaea possess numerous kinds of cytoskeletal proteins, including prokaryotic homologues of the eukaryotic actins, tubulins, and intermediate filaments, as well as other types that have been found primarily or exclusively in prokaryotes (Table 1). Understanding the diverse functions and mechanisms of the rapidly growing universe of prokaryotic cytoskeletal proteins will not only advance prokaryotic cell biology and reveal evolutionary principles, but also open up new avenues for the development of anti-microbial agents, de novo protein design, and the construction of minimal and synthetic cells.

Due to importantly beneficial effects on physical and mental health and strong association with many rehabilitation programs, Physical Activity Recognition and Measure (PARM) has been widely recognised as a key paradigm for a variety of smart healthcare applications. Traditional methods for PARM relies on designing and utilising Data fusion or machine learning techniques in processing ambient and wearable sensing data for classifying types of physical activity and removing their uncertainties. Yet they mostly focus on controlled environments with the aim of increasing types of identifiable activity subjects, improved recognition accuracy and measure robustness. The emergence of the Internet of Things (IoT) enabling technology is transferring PARM studies to an open and dynamic uncontrolled ecosystem by connecting heterogeneous cost-effective wearable devices and mobile apps and various groups of users [35] . Little is currently known about whether traditional Data fusion techniques can tackle new challenges of IoT environments and how to effectively harness and improve these technologies. In an effort to understand potential use and opportunities of Data fusion techniques in IoT enabled PARM applications, this paper will give a systematic review, critically examining PARM studies from a perspective of a novel 3D dynamic IoT based physical activity collection and validation model. It summarized traditional state-of-the-art data fusion techniques from three plane domains in the 3D dynamic IoT model: devices, persons and timeline. The paper goes on to identify some new research trends and challenges of data fusion techniques in the IoT enabled PARM studies, and discusses some key enabling techniques for tackling them.

Bakalaureusetöö Toiduainete tehnoloogia õppekaval Kali on enamasti pärmi toimel kääritatud jook, kuid varasemalt kasutati protsessis ka piimhappebaktereid (LAB). Käesoleva töö esmärk on selgitada välja, kuidas piimhappebakterid mõjutavad nii üksi kui ka koos pärmiga kaljavirde käärimist 16 tunni käärimise jooksul. Töös tehti selgeks üldine kalja tootmistehnoloogia ning võimalused tootmiseks. Vaadeldi teiste autorite tulemusi seoses kalja või pärmide/piimhappebakterite uurimisega. Valiti LAB-kultuurid: Nordwise Lactobacillus plantarum TAK69, Wildbrew Sour Pitch L. plantarum, Wildbrew Helveticus Pitch L. helveticus. Töös leiti, et happeliseim oli Sour Pitch, järgnes Helveticus Pitch ning viimaseks Nordwise. Koos pärmiga käärimisel oli suurim happesus (g/L sidrunhapet) enamasti 80/20 vahekorras LAB-pärm käärimisega ning enim sidrunhapet leidus Sour Pitchiga (2,84 g/L), veidi vähem Helveticus Pitch-iga (2,24 g/L) ning Nordwise puhul oli suurim happesus 50/50 puhul, 80/20 happesus sel puhul 1,45 g/L. 50/50 vahekorraga oli madalaim suhkrusisaldus, kuid võrreldes 80/20 vahekorraga Sour Pitch ja Helveticus Pitch puhul veidi kõrgem pH ja madalam sidrunhappe sisaldus: Sour Pitch 2,58 g/L; Helveticus Pitch 2,09 g/L; Nordwise 1,62 g/L. 100% LAB-iga käärimisel suhkrusisalduse näitaja ei langenud, pH oli kõrgeim ja sidrunhappe sisaldus madalaim, välja arvatud Sour Pitchiga, kus pH oli madalaim (3,57), kuid sidrunhappe sisaldus samuti madalaim (2,34 g/L). Poekalja pH-ga (3,45) võrreldes oli saadud kaljade pH kõrgem ning sidrunhappe sisaldus (poekaljal 1,15 g/L) samuti kõrgem. Tulemustest leidus, et kalja füüsikalis-keemilised näitajad olenevad enim kasutatavast LAB-kultuurist, väiksemal määral LAB-pärm vahekorrast juhul kui kasutati nii pärmi kui LAB-i. Ainult LAB-i kasutades oli käärimise kiirus väga aeglane ning näitajad muutusid väiksemal määral, välja arvatud Sour Pitch. Edasised uuringud ja katsed piimhappebakteritega võimaldaksid leida optimaalsed LAB-kultuurid ning meetodid hapendatud kalja tootmiseks. Kvass is a drink usually fermented with yeast but in the past lactic acid bacteria (LAB) have also been used. The purpose of this research is to monitor how LAB affect the fermentation process of kvass with or without the presence of yeast during 16 hours of fermentation. Technology and methods for kvass production were studied, along with prior research of kvass production and use of yeasts/LAB from authors. Three LABcultures were chosen: Nordwise Lactobacillus plantarum TAK69, Wildbrew Sour Pitch L. plantarum and Wildbrew Helveticus Pitch L. helveticus. In the study, Sour Pitch made for the most acidic kvass, followed by Helveticus Pitch and Nordwise was the least acidic. With the addition of yeast in fermentation, the greatest acidity (g/L citric acid) was found mostly with 80/20 proportion of LAB/yeast and the most citric acid was found with Sour Pitch (2,84 g/L), followed by Helveticus Pitch (2,24 g/L), for Nordwise, 50/50 had the highest acidity, 80/20 citric acid value was 1,45 g/L. The 50/50 proportion had the lowest sugar content but compared to 80/20, in the case of Sour Pitch and Helveticus Pitch, the pH was higher and acidity lower: Sour Pitch 2,58 g/L; Helveticus Pitch 2,09 g/L; Nordwise 1,62 g/L. In the case of 100% LAB fermentation, sugar content did not decrease, pH was the highest and acidity the lowest, except for Sour Pitch, which had the lowest pH (3,57) but also the lowest citric acid content (2,34 g/L). Compared to the pH of commercial kvass (3,45), kvass made in this research hag higher pH and also higher citric acid content (for commercial kvass 1,15 g/L). The results show, that physical-chemical indicators of kvass are most affected by the choice of LAB-cultures, less so by LAB-yeast proportions if yeast is used. When using only LAB, the speed of fermentation was slow and the indicators varied less, except for Sour Pitch. Further research and experiments with lactic acid bacteria could yield optimal LAB-cultures to use in production of acidified kvass.

Bakalaureusetöö Toiduainete tehnoloogia õppekaval Fermentereid ehk bioreaktoreid kasutatakse laialdaselt erinevates valdkondades, sealhulgas näiteks teaduslaborites mitmesuguste mikrobioloogiliste protsesside uurimiseks. Ka Eesti Maaülikooli Toiduteaduse ja toiduainete tehnoloogia õppetooli mikrobioloogia laboris on Applikon my-Control 2 L mahutavusega fermenter, millel seni puudus varasem kasutuskogemus, kuid mis omab suurt potentsiaali erinevateks rakendusteks nii mikrobioloogias kui ka toiduteaduses laiemalt. Bakalaureusetöö eesmärk on anda põhjalik ülevaade fermenterite tööpõhimõtetest, kasutusaladest ning eksperimentaalse töö alusel koostada Applikon my-Control fermenteri kasutusjuhend. Eksperimentaalse töö jaoks kasutati fermenteri riist- ja tarkvara kahe pärmikultuuri kasvatamisel, et jälgida fermentatsiooniprotsessi kulgu ja hinnata seadme töö stabiilsust. Eksperimentaalses osas jälgiti fermenteri erinevate seguri kiiruste (100-300 rpm) mõju pärmiliikide Saccharomyces pastorianus ja Saccharomyces bayanus rakkude arvule, kaalule ja kasvusöötme pH väärtusele fermentatsiooniprotsessi käigus. Leiti, et S. pastorianus’e kasvatamiseks sobisid kõik valitud segamiskiirused 100-300 rpm, kuid suurim pärmirakkude arvu muutus registreeriti segamiskiiruse 200 rpm kasutamisel (10,58 ± 0,08 log rakku/L). S. bayanus’e rakkude arvu määramine biomassi anduriga ebaõnnestus pärmi poolt moodustatud biokile tõttu, aga kaalu muutuse põhjal järeldati, et S. bayanus’e kasv toimus samuti kõige aktiivsemalt segamiskiiruse 200 rpm juures. Mõlema pärmi fermentatsiooniprotsessi käigus toimus esialgu kasvusöötme pH langus ning hiljem selle tõus. S. pastorianus kultuuri kasutamisel langes pH kuni 40. tunnini, aga S. bayanus’e fermentatsiooniprotsessi käigus langes pH minimaalseima väärtuseni juba 16 tundi pärast pärmi söötmesse lisamist. S. bayanus’e pH väärtuse muutused olid prominentsemad segamiskiiruse 300 rpm juures. Eksperimentide käigus saadud tulemused tõestasid Applikon my-Control seadme töö stabiilsust. Lisaks koostati katsete läbiviimise käigus Applikon my-Control 2 L fermenteri detailne kasutusjuhend, mis hõlbustab seadme edaspidist kasutamist erinevate katsete läbiviimisel. Fermenters, also known as bioreactors, are widely used in various fields, such as, e.g., research laboratories to investigate various microbiological processes. The laboratory of the Chair of Food Science and Technology of the Estonian University of Life Sciences has a new Applikon my-Control fermenter, have so far not had any user-experience by stuff, but has great potential for various applications in microbiology and food science in general. The aim of this Bachelor's thesis is to provide a thorough overview of the operating principles and applications of fermenters in general and to compile the operating manual for the Applikon my-Control fermenter based on the experiments. For experimental work, current fermenter hardware and software technologies were used to grow two types of yeast, monitor the progress of the fermentation process and assess the stability of the equipment. In the experimental part, the effect of different stirrer agitation speeds (100-300 rpm) on the number of cells of Saccharomyces pastorianus and Saccharomyces bayanus, their weight and the overall pH of the growth medium were evaluated during the fermentation process. Stirring speeds of 100-300 rpm were found to be suitable for growing S. pastorianus, but the largest change in the number of yeast cells was recorded at a stirring speed of 200 rpm (10.58 ± 0.08 log cells/L). S. bayanus cell counting with the biomass sensor failed, however, due to its biofilm forming ability, but based on the changes in weight, it was determined that S. bayanus growth was also most active at a stirring speed of 200 rpm. During the fermentation process of both types of yeast, the pH of the growth medium initially decreased and later increased. During S. pastorianus growth, the pH was decreasing until the 40-hour mark, but during the fermentation process of S. bayanus, the pH dropped to a minimum as early as 16 hours after the addition of the yeast to the medium. Changes in the pH of S. bayanus were more prominent at an increased stirring speed of 300 rpm. The results of the experiments showed the stability of the Applikon my-Control. In addition, a detailed user manual for the Applikon my-Control was compiled in order for the device to be used for various continual tests.

Saline stress is commonly encountered by microbial cell factories. The molecular mechanisms of saline stress are identified in the conventional budding yeast Saccharomyces cerevisiae that is used in biotechnology. However, for diversification of microbial cell factories, non-conventional yeasts provide an attractive opportunity but saline stress in these yeasts is not very well investigated. This thesis is focused on achieving foundational understanding of morphological and physiological response to saline stress of conventional and non-conventional yeasts by comparing Saccharomyces cerevisiae W303 and CEN.PK113-7D together with Kluyveromyces marxianus and Rhodotorula toruloides. The results showed that effects of Na+ stress could be ameliorated by potassium supplementation in S. cerevisiae CEN.PK113-7D increasing its specific growth rate by almost four-fold. In addition, combination of sodium and potassium salts resulted in impaired acetic acid and/or ethanol metabolism in S. cerevisiae. Further, in this thesis, a neural network-based image analysis software pipeline for a quantitative evaluation of saline stress response was developed that could be used to determine differences in cell volume among the strains. Quantitative image analysis found a high variability in cell volume dynamics implying distinct stress response mechanisms among different yeasts. The cell volume differences could be used to model saline stress responses in various bioprocesses in biotechnology. In estonian: Kõrgete soola kontsenteratsioonide poolt põhjustatud osmootne stress on levinud stressifaktor tööstuslikult kasutavatele mikroorganismidel põhinevatele rakuvabrikutele. Soola stressi molekulaarsed mehhanismid on tuvastatud tavapärases punguvas pärmis Saccharomyces cerevisiae, mida kasutatakse biotehnoloogias. Mittekonventsionaalsed pärmid kujundavad endast atraktiivset võimalust mikrobioloogiliste rakuvabrikute diversifitseerimiseks, kuid soola stress sellistes pärmides on tänini praktiliselt kirjeldamata. Käesoleva bakalaureusetöö eesmärk on fundamentaalse arusaama saavutamine konventsionaalsete ja mittekonventsionaalsete pärmide ja nende soola stressi morfoloogilise ja füsioloogilise vastuste suhtes. Võrdluseks on kasutatud Saccharomyces cerevisiae CEN.PK113-7D ja W303, Kluyveromyces marxianus ja Rhodotorula toruloides pärmide tüvesid. Tulemused näidavad, et naatriumi tingitud stressi effektid võib leevendada kaaliumi lisamisega pärmis S. cerevisiae CEN.PK113-7D, peaaegu neljakordistades tema erikasvukiirust. Lisaks, naatriumi ja naatriumi/kaaliumi kombinatsioonid põhjustasid nõrgenenud atsetaadi ja/või etanooli metabolismi pärmis S. cerevisiae. Peale selle, tehisnärvivõrgul põhinev pilditöötluse tarkvara oli välja töötatud soola stressist põhjustatud rakkude ruumala muutuste kvantitatiivseks analüüsiks. Kvantitatiivne piltide analüüs on leidnud suurt variatiivsust rakkude mahus, vihjates eristuvatele stressile vastuste mehhanismidele erinevates pärmides. Rakkude mahu erinevusi võib kasutada soola stressile vastuste modelleerimiseks mitmesugustes biotehnoloogia bioprotsessides.

Purpose Gastroesophageal reflux after surgical repair of esophageal atresia (EA) can be associated with complications, such as esophageal stricture. Recent guidelines recommend prophylactic anti-reflux medication (PARM) after EA repair. However, the effectiveness of PARM is still unclear. The aim of this study was to review evidence surrounding the use of PARM in children operated for EA. Methods We performed a systematic review and meta-analysis. We searched Medline, EMBASE, and the Cochrane Databases from inception until the end of 2016 for comparative studies of PARM versus no PARM (control). Primary outcome was postoperative esophageal stricture. Quality of evidence was assessed using GRADE system. Results We identified four observational studies that focused on esophageal stricture as an outcome. A total of 362 patients were included in meta-analysis. There was no significant difference in esophageal stricture rates between PARM and control (OR = 1.14; 95% CI = 0.61–2.13; p = 0.68; I2 = 38%). The quality of the evidence was very low, due to lack of precision as a consequence of small study sizes. Conclusions Our results indicate that PARM does not reduce the incidence of esophageal stricture after EA repair. Future well-controlled prospective studies are needed to obtain higher quality evidence.

Clostridium perfringens produces an extensive repertoire of toxins and extracellular enzymes, many of which are intimately involved in the progression of disease and are encoded by genes on conjugative plasmids. In addition, many C. perfringens strains can carry up to five of these conjugative toxin or antimicrobial resistance plasmids, each of which has a similar 35kb backbone. This conserved backbone includes the tcp conjugation locus and the central control region (CCR), which encodes genes involved in plasmid regulation, replication and partitioning, including a parMRC partitioning locus. Most conjugative plasmids in C. perfringens have a conserved replication protein, raising questions as to how multiple, closely related plasmids are maintained within a single strain. Bioinformatics analysis has highlighted the presence of at least 10 different parMRC partitioning system families (parMRCA-J) in these plasmids, with differences in amino acid sequence identity between each ParM family ranging from 15% to 54%. No two plasmids that encode genes belonging to the same partitioning family have been observed in a single strain, suggesting that these families represent the basis for plasmid incompatibility. In an attempt to validate the proposed parMRC incompatibility groups, genetically marked C. perfringens plasmids encoding identical parMRCC or parMRCD homologues or different combinations of parMRCA, parMRCC and parMRCD family homologues were introduced into a single strain via conjugation. The stability of each plasmid was determined using an incompatibility assay in which the plasmid profile of each strain was monitored over the course of two days in the absence of direct selection. The results showed that plasmids with identical parMRCC or parMRCD homologues were incompatible and could not coexist in the absence of external selection. By contrast, plasmids that encoded different parMRC homologues were compatible and could coexist in the same cell in the absence of selection, with the exception of strains housing parMRCC and parMRCD combinations, which showed a minor incompatibility phenotype. In conclusion, we have provided the first direct evidence of plasmid incompatibility in Clostridium spp. and have shown experimentally that the compatibility of conjugative C. perfringens plasmids correlates with the presence of parMRC-like partitioning systems of different phylogenetic subfamilies.

Microfilaments (actin) and microtubules represent the extremes in eukaryotic cytoskeleton cross-sectional dimensions, raising the question of whether filament architectures are limited by protein fold. Here, we report the cryoelectron microscopy structure of a complex filament formed from 15 protofilaments of an actin-like protein. This actin-like ParM is encoded on the large pCBH Clostridium botulinum plasmid. In cross-section, the ~26 nm diameter filament comprises a central helical protofilament surrounded by intermediate and outer layers of six and eight twisted protofilaments, respectively. Alternating polarity of the layers allows for similar lateral contacts between each layer. This filament design is stiffer than the actin filament, and has likely been selected for during evolution to move large cargos. The comparable sizes of microtubule and pCBH ParM filaments indicate that larger filament architectures are not limited by the protomer fold. Instead, function appears to have been the evolutionary driving force to produce broad, complex filaments., The plasmid-segregating actin-like protein ParM is encoded on the large, toxin carrying plasmid pCBH from Clostridium botulinum. Here the authors present the cryo-EM structure of the ParM filament that is formed from the association of 15 protofilaments and discuss its architecture.

PHOX2B 20/27 polyalanine repeat mutation (PARM) in patients with congenital central hypoventilation syndrome (CCHS) is generally associated with full-time ventilator dependence, Hirschsprung disease, and increased risk for cardiac asystole. We follow a 14-year-old boy with CCHS PHOX2B 20/27 PARM who is full-time ventilator dependent via tracheostomy and has Hirschsprung disease. His mother, age 52 years, has a history of prolonged recovery from anesthesia and an elevated serum bicarbonate level of 45 mEq/L discovered on routine blood chemistry. PHOX2B gene mutation analysis was performed and showed an identical 20/27 PARM, diagnostic of CCHS. Late-onset CCHS has been reported in those with 20/24, 20/25 PHOX2B PARM, and in nonpolyalanine repeat mutations. This is the first report of a patient with PHOX2B 20/27 PARM with a mild phenotype diagnosed during adulthood. This unusual presentation supports the screening for PHOX2B mutations in parents of children with CCHS. CITATION: Kasi AS, Kun SS, Keens TG, Perez IA. Adult with PHOX2B mutation and late-onset congenital central hypoventilation syndrome. J Clin Sleep Med. 2018;14(12):2079–2081.

Plasmids that encode the same replication machinery are generally unable to coexist in the same bacterial cell. However,Clostridium perfringensstrains often carry multiple conjugative toxin or antibiotic resistance plasmids that are closely related and encode similar Rep proteins. In many bacteria, plasmid partitioning upon cell division involves a ParMRC system and there are ~10 different ParMRC families inC. perfringens, with differences in amino acid sequences between each ParM family (15% − 54% identity). Since plasmids encoding genes belonging to the same ParMRC family are not observed in the same strain, these families appear to represent the basis for plasmid compatibility inC. perfringens. To understand this process, we examined the key recognition steps between ParR DNA-binding proteins and theirparCbinding sites. The ParR proteins bound to sequences within aparCsite from the same ParMRC family, but could not interact with aparCsite from a different ParMRC family. These data provide evidence that compatibility of the conjugative toxin plasmids ofC. perfringensis mediated by theirparMRC-like partitioning systems. This process provides a selective advantage by enabling the host bacterium to maintain separate plasmids that encode toxins that are specific for different host targets.

Magnetotactic bacteria (MTB), a group of phylogenetically diverse organisms that use their unique intracellular magnetosome organelles to swim along the Earth’s magnetic field, play important roles in the biogeochemical cycles of iron and sulfur. Previous studies have revealed that the bacterial actin protein MamK plays essential roles in the linear arrangement of magnetosomes in MTB cells belonging to the Proteobacteria phylum. However, the molecular mechanisms of multiple-magnetosome-chain arrangements in MTB remain largely unknown. Here, we report that the MamK filaments from the uncultivated ‘Candidatus Magnetobacterium casensis’ (Mcas) within the phylum Nitrospirae polymerized in the presence of ATP alone and were stable without obvious ATP hydrolysis-mediated disassembly. MamK in Mcas can convert NTP to NDP and NDP to NMP, showing the highest preference to ATP. Unlike its Magnetospirillum counterparts, which form a single magnetosome chain, or other bacterial actins such as MreB and ParM, the polymerized MamK from Mcas is independent of metal ions and nucleotides except for ATP, and is assembled into well-ordered filamentous bundles consisted of multiple filaments. Our results suggest a dynamically stable assembly of MamK from the uncultivated Nitrospirae MTB that synthesizes multiple magnetosome chains per cell. These findings further improve the current knowledge of biomineralization and organelle biogenesis in prokaryotic systems. Electronic supplementary material The online version of this article (doi:10.1007/s13238-016-0253-x) contains supplementary material, which is available to authorized users.

Due to importantly beneficial effects on physical and mental health and strong association with many rehabilitation programs, Physical Activity Recognition and Monitoring (PARM) have been considered as a key paradigm for smart healthcare. Traditional methods for PARM focus on controlled environments with the aim of increasing the types of identifiable activity subjects complete and improving recognition accuracy and system robustness by means of novel body-worn sensors or advanced learning algorithms. The emergence of the Internet of Things (IoT) enabling technology is transferring PARM studies to open and connected uncontrolled environments by connecting heterogeneous cost-effective wearable devices and mobile apps. Little is currently known about whether traditional PARM technologies can tackle the new challenges of IoT environments and how to effectively harness and improve these technologies. In an effort to understand the use of IoT technologies in PARM studies, this paper will give a systematic review, critically examining PARM studies from a typical IoT layer-based perspective. It will firstly summarize the state-of-the-art in traditional PARM methodologies as used in the healthcare domain, including sensory, feature extraction and recognition techniques. The paper goes on to identify some new research trends and challenges of PARM studies in the IoT environments, and discusses some key enabling techniques for tackling them. Finally, this paper consider some of the successful case studies in the area and look at the possible future industrial applications of PARM in smart healthcare.

Low copy-number plasmid pLS32 of Bacillus subtilis subsp. natto contains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB, and the centromeric sequence parN Similar to the ParMRC partitioning system from Escherichia coli plasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids through interactions with AlfB and parN Because, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of nonbundling AlfA filaments to 3.4-A resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin fold has been accommodated by unique longitudinal and lateral contacts, while still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments, we obtained a pseudoatomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly antiphasic with near eightfold helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, how deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerization, nucleotide hydrolysis, and antiparallel doublet formation are retained to fulfill the system's biological raison d'etre.

Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 A resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. Abstract Parents are important role models for their children's eating behaviours. This study aimed to further validate the recently developed Parental Modelling of Eating Behaviours Scale (PARM) by examining the relationships between maternal self-reports on the PARM with the modelling practices exhibited by these mothers during three family mealtime observations. Relationships between observed maternal modelling and maternal reports of children's eating behaviours were also explored. Seventeen mothers with children aged between 2 and 6 years were video recorded at home on three separate occasions whilst eating a meal with their child. Mothers also completed the PARM, the Children's Eating Behaviour Questionnaire and provided demographic information about themselves and their child. Findings provided validation for all three PARM subscales, which were positively associated with their observed counterparts on the observational coding scheme (PARM-O). The results also indicate that habituation to observations did not change the feeding behaviours displayed by mothers. In addition, observed maternal modelling was significantly related to children's food responsiveness (i.e., their interest in and desire for foods), enjoyment of food, and food fussiness. This study makes three important contributions to the literature. It provides construct validation for the PARM measure and provides further observational support for maternal modelling being related to lower levels of food fussiness and higher levels of food enjoyment in their children. These findings also suggest that maternal feeding behaviours remain consistent across repeated observations of family mealtimes, providing validation for previous research which has used single observations.

In the present paper, a method by which to suppress the temperature increase in pneumatic artificial rubber muscles is proposed. The pneumatic artificial rubber muscle (PARM) is a flexible actuator. The use of PARMs in industrial and scientific applications has increased significantly as a result of their advantages. Usually, compressed air is charged or discharged from one end of the PARM as it is inflated or deflated. The PARM expands radially and contracts axially when inflated. The closed end is referred to herein as the end part. Since the fluidity of air at the end part of the PARM is low, when a PARM is pressurized repeatedly, the temperature at the end part increases significantly. This temperature increase shortens the operating life of the PARM and limits the characteristics of continuous drive. A method of addressing this temperature increase is proposed herein. A pipeline is installed in the PARM, and compressed air is charged or discharged through the pipeline in order to promote the convection of air at the end part of the PARM. As a result, the temperature increase in the PARM is suppressed. We confirmed experimentally that the proposed method can effectively suppress the temperature increase in the PARM.

One of the well-known functions of the bacterial cytoskeleton is plasmid segregation. Type II plasmid segregation systems, among the best characterized with respect to the mechanism of action, possess an actin-like cytomotive filament as the motor component. This chapter describes the essential components of the plasmid segregation machinery and their mechanism of action, concentrating on the actin-like protein family of the bacterial cytoskeleton. The structures of the actin-like filaments depend on their nucleotide state and these in turn contribute to the dynamics of the filaments. The components that link the filaments to the plasmid DNA also regulate filament dynamics. The modulation of the dynamics facilitates the cytomotive filament to function as a mitotic spindle with a minimal number of components.

Bipolar elongation of filaments of the bacterial actin homolog ParM drives movement of newly replicated plasmid DNA to opposite poles of a bacterial cell. We used a combination of vitreous sectioning and electron cryotomography to study this DNA partitioning system directly in native, frozen cells. The diffraction patterns from overexpressed ParM bundles in electron cryotomographic reconstructions were used to unambiguously identify ParM filaments in Escherichia coli cells. Using a low–copy number plasmid encoding components required for partitioning, we observed small bundles of three to five intracellular ParM filaments that were situated close to the edge of the nucleoid. We propose that this may indicate the capture of plasmid DNA within the periphery of this loosely defined, chromosome-containing region.

Bacterial conjugation is a form of type IV secretion used to transport protein and DNA directly to recipient bacteria. The process is cell contact-dependent, yet the mechanisms enabling extracellular events to trigger plasmid transfer to begin inside the cell remain obscure. In this study of plasmid R1 we investigated the role of plasmid proteins in the initiation of gene transfer. We find that TraI, the central regulator of conjugative DNA processing, interacts physically and functionally with the plasmid partitioning proteins ParM and ParR. These interactions stimulate TraI catalyzed relaxation of plasmid DNA in vivo and in vitro and increase ParM ATPase activity. ParM also binds the coupling protein TraD and VirB4-like channel ATPase TraC. Together, these protein-protein interactions probably act to co-localize the transfer components intracellularly and promote assembly of the conjugation machinery. Importantly these data also indicate that the continued association of ParM and ParR at the conjugative pore is necessary for plasmid transfer to start efficiently. Moreover, the conjugative pilus and underlying secretion machinery assembled in the absence of Par proteins mediate poor biofilm formation and are completely dysfunctional for pilus specific R17 bacteriophage uptake. Thus, functional integration of Par components at the interface of relaxosome, coupling protein and channel ATPases appears important for an optimal conformation and effective activation of the transfer machinery. We conclude that low copy plasmid R1 has evolved an active segregation system that optimizes both its vertical and lateral modes of dissemination.

Väitekirja elektrooniline versioon ei sisalda publikatsioone., Kõikides eukarüootsetes rakkudes viib valke kodeerivate geenide transkriptsiooni läbi RNA polümeraas II (RNAPII). Protsessi, mille käigus sünteesitakse mRNA, nimetatakse transkriptsioonitsükliks ning see tsükkel jaotatakse kolme etappi – transkriptsiooni initsiatsioon, elongatsioon ning terminatsioon. Käesolevas töös uurisin RNAPII elongatsiooni mehhanisme, kasutades mudelorganismina pagaripärmi Saccharomyces cerevisiae. Antud töö esmaseks eesmärgiks oli tekitada kõrgel tasemel transkribeeritava mudelgeeni sisse nn. vaigistavate valgukomplekside poolt heterokromatiinne ala ning uurida selle struktuuri mõju transkriptsiooni läbi viivale RNAPII kompleksile. Selgus, et elongatsiooni etapis olev RNAPII suudab transkribeerida läbi heterokromatiini ning see viib vaigistavate valgukomplekside eemaldamisele. Lisaks selgus, et heterokromatiini läbimiseks RNAPII poolt on vajalik histoon H3 56. positsioonis oleva lüsiinijäägi atsetüleerimine. Teiseks huvipakkuvaks küsimuseks oli, mis juhtub siis, kui kõrgel tasemel transkribeeritavasse mudelgeeni viia sisse replikatsiooni algusala (origin), millel moodustuvad pre-replikatiivsed kompleksid. Nägime, et elongatsiooni etapis olev RNAPII suudab eemaldada kodeerivas alas moodustunud pre-replikatiivsed kompleksid ilma, et selline kokkupuude häiriks toimuvat transkriptsiooniprotsessi. Lisaks taastatakse pärast transkriptsiooni lõppemist pre-replikatiivsed kompleksid inaktiveeritud replikatsiooni algusaladel ning sellised uuesti moodustunud kompleksid on järgnevas S-faasis funktsionaalsed. Lõpetuseks, eelnevates, terveid rakupopulatsioone hõlmavates katsetes oli näidatud, et RNAPII kompleksid jaotusid geenil ebaühtlaselt. Sellest tulenevalt oli käesoleva töö kolmandaks eesmärgiks uurida RNAPII komplekside jaotumist kõrgelt transkribeeritaval mudelgeenil ühe raku tasemel. Vastupidiselt eelnevatele tulemustele leidsime, et RNAPII kompleksid jaotusid ühtlaselt kogu geeni ulatuses., Transcription of all eukaryotic protein-coding genes is carried out by RNA polymerase II (RNAPII). The process of mRNA synthesis is called transcription cycle and this cycle is divided into three steps – initiation, elongation and termination. The current thesis focuses on mechanisms of RNAPII transcription elongation in budding yeast Saccharomyces cerevisiae. The first goal of this thesis was to introduce repressive heterochromatin into the highly transcribed model gene and to analyze how this structure influences elongating RNAPII. Our results show that elongating RNAPII can transcribe through heterochromatin, leading to displacement of silencing complexes. In addition, acetylation of H3K56 is essential for RNAPII elongation through heterochromatin. The second interesting question was what is the outcome of elongating RNAPII encountering pre-replicative complexes formed on replication origin that lies in the middle of the highly transcribed model gene. We determined that pre-replicative complexes formed on replication origins in coding region are removed by elongating RNAPII and this encountering does not interfere with RNAPII transcription elongation. Additionally, transcription-coupled inactivation of replication origins is reversible by re-assembling pre-replicative complexes when transcription stops and these newly formed complexes are fully functional in the following S-phase. Finally, in previous studies averaging RNAPII density from whole population of cells, uneven distribution of RNAPII has been reported. Therefore the third goal of this thesis was to determine the distribution of RNAPII on highly transcribed model gene on single cell level. Intriguingly, our results demonstrate that RNAPII complexes are distributed uniformly throughout the entire length of the gene.

Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments.

Significance Actins and tubulins have dedicated functions that vary between eukaryotes and prokaryotes. During cell division, the prokaryotic contractile ring depends on the tubulin-like protein FtsZ, whereas this task relies on actin in eukaryotes. In contrast, microtubules orchestrate DNA segregation in eukaryotes, yet prokaryotic plasmid segregation often depends on actin-like proteins; this implies that actins and tubulins have somewhat interchangeable properties. Hence, we sought a bacterial filament that more closely resembles microtubules. Here, we report an actin from Bacillus thuringiensis that forms dynamic, antiparallel, two-stranded supercoiled filaments, which pair in the presence of a binding partner to form hollow cylinders. Thus, in this prokaryote, the actin fold has evolved to produce a filament system with comparable properties to the eukaryotic microtubule.

Bacterial actins polymerize in the presence of nucleotide (preferably ATP), form a common arrangement of monomeric interfaces within a protofilament, and undergo ATP hydrolysis-dependent change in stability of the filament—all of which contribute to performing their respective functions. The relative stability of the filament in the ADP-bound form compared to that of ATP and the rate of addition of monomers at the two ends decide the filament dynamics. One of the major differences between eukaryotic actin and bacterial actins is the variety in protofilament arrangements and dynamics exhibited by the latter. The filament structure and the polymerization dynamics enable them to perform various functions such as shape determination in rod-shaped bacteria (MreB), cell division (FtsA), plasmid segregation (ParM family of actin-like proteins), and organelle positioning (MamK). Though the architecture and dynamics of a few representative filaments have been studied, information on the effect of interacting partners on bacterial actin filament dynamics is not very well known. The chapter reviews some of the structural and functional aspects of bacterial actins, with special focus on the effect that interacting partners exert on the dynamics of bacterial actins, and how these assist them to carry out the functions within the bacterial cell.

Many pathogenic strains of Clostridium perfringens carry several highly similar toxin or antibiotic resistance plasmids that have 35 to 40 kb of very closely related syntenous sequences, including regions that carry the genes encoding conjugative transfer, plasmid replication and plasmid maintenance functions. Key questions are how are these closely related plasmids stably maintained in the same cell and what is the basis for plasmid incompatibility in C. perfringens. Comparative analysis of the Rep proteins encoded by these plasmids suggested that this protein was not the basis for plasmid incompatibility since plasmids carried in a single strain often encoded an almost identical Rep protein. These plasmids all carried a similar, but not identical, parMRC plasmid partitioning locus. Phylogenetic analysis of the deduced ParM proteins revealed that these proteins could be divided into ten separate groups. Importantly, in every strain that carried more than one of these plasmids, the respective ParM proteins were from different phylogenetic groups. Similar observations were made from the analysis of phylogenetic trees of the ParR proteins and the parC loci. These findings provide evidence that the basis for plasmid incompatibility in the conjugative toxin and resistance plasmid family from C. perfringens resides in subtle differences in the parMRC plasmid partitioning loci carried by these plasmids.

Background Clinical practice guidelines need to be regularly updated with current literature in order to remain relevant. This paper reports on the approach taken by the Philippine Academy of Rehabilitation Medicine (PARM). This dovetails with its writing guide, which underpinned its foundational work in contextualizing guidelines for stroke and low back pain (LBP) in 2011. Methods Working groups of Filipino rehabilitation physicians and allied health practitioners met to reconsider and modify, where indicated, the ‘typical’ Filipino patient care pathways established in the foundation guidelines. New clinical guidelines on stroke and low back pain which had been published internationally in the last 3 years were identified using a search of electronic databases. The methodological quality of each guideline was assessed using the iCAHE Guideline Quality Checklist, and only those guidelines which provided full text references, evidence hierarchy and quality appraisal of the included literature, were included in the PARM update. Each of the PARM-endorsed recommendations was then reviewed, in light of new literature presented in the included clinical guidelines. A novel standard updating approach was developed based on the criteria reported by Johnston et al. (Int J Technol Assess Health Care 19(4):646–655, 2003) and then modified to incorporate wording from the foundational PARM writing guide. The new updating tool was debated, pilot-tested and agreed upon by the PARM working groups, before being applied to the guideline updating process. Results Ten new guidelines on stroke and eleven for low back pain were identified. Guideline quality scores were moderate to good, however not all guidelines comprehensively linked the evidence body underpinning recommendations with the literature. Consequently only five stroke and four low back pain guidelines were included. The modified PARM updating guide was applied by all working groups to ensure standardization of the wording of updated recommendations and the underpinning evidence bases. Conclusions The updating tool provides a simple, standard and novel approach that incorporates evidence hierarchy and quality, and wordings of recommendations. It could be used efficiently by other guideline updaters particularly in developing countries, where resources for guideline development and updates are limited. When many people are involved in guideline writing, there is always the possibility of ‘slippage’ in use of wording and interpretation of evidence. The PARM updating tool provides a mechanism for maintaining a standard process for guideline updating processes that can be followed by clinicians with basic training in evidence-based practice principles. Electronic supplementary material The online version of this article (doi:10.1186/s13104-015-1588-8) contains supplementary material, which is available to authorized users.

From yeast to man, an evolutionary distance of 1.3 billion years, the F-actin filament structure has been conserved largely in line with the 94% sequence identity. The situation is entirely different in bacteria. In comparison to eukaryotic actins, the bacterial actin-like proteins (ALPs) show medium to low levels of sequence identity. This is extreme in the case of the ParM family of proteins, which often display less than 20% identity. ParMs are plasmid segregation proteins that form the polymerizing motors that propel pairs of plasmids to the extremities of a cell prior to cell division, ensuring faithful inheritance of the plasmid. Recently, exotic ParM filament structures have been elucidated that show ParM filament geometries are not limited to the standard polar pair of strands typified by actin. Four-stranded non-polar ParM filaments existing as open or closed nanotubules are found in Clostridium tetani and Bacillus thuringiensis, respectively. These diverse architectures indicate that the actin fold is capable of forming a large variety of filament morphologies, and that the conception of the “actin” filament has been heavily influenced by its conservation in eukaryotes. Here, we review the history of the structure determination of the eukaryotic actin filament to give a sense of context for the discovery of the new ParM filament structures. We describe the novel ParM geometries and predict that even more complex actin-like filaments may exist in bacteria. Finally, we compare the architectures of filaments arising from the actin and tubulin folds and conclude that the basic units possess similar properties that can each form a range of structures. Thus, the use of the actin fold in microfilaments and the tubulin fold for microtubules likely arose from a wider range of filament possibilities, but became entrenched as those architectures in early eukaryotes. Published version