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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.

The ParM protein, encoded by the parMRC locus from plasmid R1, is the best-characterized actin homolog present in prokaryotes. In conjunction with a nucleoprotein complex formed by a DNA binding protein ParR and a DNA centromere parC, ParM constructs a simple mitotic spindle that pushes plasmid DNA to opposite poles of a bacterial cell for DNA segregation. Like actin, ParM assembles into ATP-dependent double-stranded, helical filaments via a nucleation condensation reaction. However, unlike actin, it displays three kinetic properties that are essential to its function: rapid spontaneous nucleation, bilateral elongation and ATPase dependent dynamic instability: the stochastic switching between filament elongation and rapid catastrophic shrinkage. In this dissertation, we investigated three questions related to ParM's evolution and function. First, many divergent R1 ParM homologs are encoded by other plasmids, and function in plasmid DNA segregation. We asked whether the filament architecture and assembly dynamics are conserved in a divergent member of the ParM family, the ParM protein from plasmid pB171. Using microscopic and biophysical techniques, we compared and contrasted the architecture and assembly of these related proteins. Despite being only 41% identical, we find that R1 and pB171 ParM polymerize into nearly identical filaments that display similar assembly dynamics, suggesting that structure and function are conserved in the ParM family. Second, we asked whether ParM prefers ATP or GTP in vitro. Using biochemical assays and TIRF microscopy, we find that ParM displays similar polymerization kinetics and stability in the presence of either nucleotide, but binds to ATP ten times tighter than GTP. Third, the structural basis for ParM filament dynamic instability remains poorly understood. Using cryo-electron microscopy to evaluate ParM filaments, we find that the nucleotide binding pocket of ParM protomers exist in an open or closed state, and that ATP hydrolysis shifts the distribution of protomers states; these findings suggest a mechanism for dynamic instability.

The par operon of the R1 plasmid constructs a DNA segregating spindle from three components, parC, ParR, and ParM. parC is a centromeric stretch of DNA which is bound by the protein ParR. ParM is an actin homologue that polymerizes into two stranded filaments that can interact with the ParR/parC complex. These three components are both necessary and sufficient to segregate parC containing plasmids, implying that the par operon is a biochemically self-contained DNA segregation apparatus. To elucidate how this system functions to segregate plasmid DNA, we wished to determine the intrinsic kinetic properties of ParM assembly, and how this assembly is regulated by the interaction with its cargo, the ParR/parC complex.We find that ParM displays three properties that distinguish its assembly from that of eukaryotic actins. 1) ParM displays hydrolysis dependent dynamic instability, the stochastic switching between states of growth and rapid depolymerization. 2) ParM filaments display a very rapid rate of nucleation, occurring at a rate 200-fold faster than actin. 3) ParM filaments exhibit no kinetic polarity, with equal rates of growth at each filament end.By combining ParM with bead immobilized ParR/parC we demonstrate that these three components can create bipolar spindles in vitro similar to those observed in vivo. ParR/parC coated microspheres are connected by bundles of ParM filaments that elongate over time, pushing the microspheres apart over long distances. Speckle microscopy and photobleaching experiments demonstrate that this motility is driven by insertional polymerization at the bead surface. We find that the ParR/parC complex prevents ParM filament ends from catastrophe by stabilizing filaments down to the ATP-ParM critical concentration. Futhermore, we show that the dynamic instability of ParM is critical to the spatial regulation of the R1 spindle, as filament ends that are not bound at each end by ParR/parC are destined to undergo catastrophe. The dynamic instability of ParM also provides an energetic differential to power spindle elongation, as the turnover of background, unbound filaments provide the monomer excess required for the growth of stabilized, spindle associated filaments.

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.