Characterization of in vitro chlamydial cultures in low-oxygen atmospheres

To mimic in vivo conditions during chlamydial infections, Chlamydia trachomatis serovar D and Chlamydia pneumoniae CWL029 were cultured in low-oxygen atmospheres containing 4% O(2), with parallel controls cultured in atmospheric air. Both were enriched with 5% CO(2). The results showed a dramatic increase in the growth of C. pneumoniae but not of C. trachomatis.     

Metal Binding in Photosystem II Super- and Subcomplexes from Barley Thylakoids

Metals exert important functions in the chloroplast of plants, where they act as cofactors and catalysts in the photosynthetic electron transport chain. In particular, manganese (Mn) has a key function because of its indispensable role in the water-splitting reaction of photosystem II (PSII). More and better knowledge is required on how the various complexes of PSII are affected in response to, for example, nutritional disorders and other environmental stress conditions. We here present, to our knowledge, a new method that allows the analysis of metal binding in intact photosynthetic complexes of barley (Hordeum vulgare) thylakoids. The method is based on size exclusion chromatography coupled to inductively coupled plasma triple-quadrupole mass spectrometry. Proper fractionation of PSII super- and subcomplexes was achieved by critical selection of elution buffers, detergents for protein solubilization, and stabilizers to maintain complex integrity. The applicability of the method was shown by quantification of Mn binding in PSII from thylakoids of two barley genotypes with contrasting Mn efficiency exposed to increasing levels of Mn deficiency. The amount of PSII supercomplexes was drastically reduced in response to Mn deficiency. The Mn efficient genotype bound significantly more Mn per unit of PSII under control and mild Mn deficiency conditions than the inefficient genotype, despite having lower or similar total leaf Mn concentrations. It is concluded that the new method facilitates studies of the internal use of Mn and other biometals in various PSII complexes as well as their relative dynamics according to changes in environmental conditions.Several metals are important for chloroplast functioning, particularly in the photosynthetic apparatus, where they act as cofactors and catalysts in electron transport processes (Merchant, 2006; Nouet et al., 2011; Yruela, 2013). The photosynthetic biometals include iron (Fe) in the form of Fe-S clusters in PSI, heme-bridged Fe (cytochrome b₅₅₉) and nonheme Fe in PSII, copper (Cu) in plastocyanin, magnesium (Mg) in chlorophyll (Chl), and calcium (Ca) and manganese (Mn) in PSII. Mn has a very special role because a metal cluster of four Mn ions and one Ca ion comprises the catalytic center of the oxygen evolving complex (OEC) in PSII (Ono et al., 1992; Umena et al., 2011). In the OEC, water is split, and molecular oxygen is produced by the photosynthetic light reactions. The photosynthetic biometals are, however, highly reactive and involved in a multitude of side reactions, which constitute a challenge for metal homeostasis. Accordingly, the handling of metals must be tightly regulated, and they must be kept within specific concentration ranges inside living cells to ensure adequate supply, while at the same time, avoiding oxidative stress (Pakrasi et al., 2001; Shcolnick and Keren, 2006; Møller et al., 2007).PSII is a large pigment-protein complex localized in the grana regions of the thylakoid membrane of chloroplasts. The basic structure of PSII is a monomer, and each complex contains more than 40 different proteins bound either stably or transiently (Nelson and Yocum, 2006; Shi et al., 2012; Järvi et al., 2015). The luminal surfaces of PSII are associated with the extrinsic proteins PsbO, PsbP, and PsbQ, which shield and support the catalytic Mn cluster and are required for efficient oxygen evolution (Roose et al., 2007; Bricker et al., 2012; Liu et al., 2014). After dimerization of the monomer, the complex associates with multiple copies of the light-harvesting antenna complex II (LHCII), forming various types of functional PSII-LHCII supercomplexes (Tikkanen et al., 2008; Kouřil et al., 2012; Shi et al., 2012).Intact PSII-LHCII supercomplexes have been successfully isolated, characterized, and refined from, for example, pea (Pisum sativum; Barera et al., 2012), Arabidopsis (Arabidopsis thaliana; Caffarri et al., 2009), and green algae (Chlamydomonas reinhardtii; Tokutsu et al., 2012). The procedure has typically involved Suc density gradient ultracentrifugation. Also, blue native (BN)-PAGE has been optimized for the separation and proteomic characterization of thylakoid PSII-LHCII supercomplexes (Heinemeyer et al., 2004; Järvi et al., 2011; Pagliano et al., 2014). The supramolecular organization of isolated PSII is very much dependent on the choice of detergent for efficient solubilization of the membrane-bound photosynthetic pigment-protein complexes. In recent years, dodecyl maltoside (DM) has become a commonly used detergent for one-step isolation of integral membrane proteins and complexes from thylakoids (Eshaghi et al., 1999; van Roon et al., 2000; Dekker et al., 2002; Pagliano et al., 2011). This detergent exists in two isomeric forms (α-DM and β-DM), of which α-DM is a milder detergent than β-DM, thereby better preserving the integrity of large PSII-LHCII supercomplexes (Pagliano et al., 2012).The major challenge associated with purification of higher plant PSII-LHCII supercomplexes is to obtain and subsequently, maintain the integrity of PSII super- and subcomplexes, including cofactors and the extrinsic proteins. To prevent dissociation of biometals and the extrinsic proteins from PSII, the osmoprotectant betaine (Papageorgiou et al., 1991; Papageorgiou and Murata, 1995) has successfully been included in the buffer of Suc gradients (Boekema et al., 1998; Tokutsu et al., 2012). Although the above-mentioned methods primarily have focused on the characterization and structural organization of isolated PSII-LHCII supercomplexes, no bench-top method has been available that allows direct analysis of the actual metal binding in PSII super- and subcomplexes. Such a method is required in order to fully understand how Mn and other photosynthetic biometals interact with the photosynthetic complexes, in particular PSII, and how the metal binding affects PSII dynamics under changing environmental conditions, including plant nutritional disorders.We here present a robust and highly sensitive method for analysis of metal binding in PSII-LHCII super- and subcomplexes from isolated barley (Hordeum vulgare) thylakoids. The method is based on size exclusion chromatography (SEC) coupled to inductively coupled plasma (ICP) triple-quadrupole (QQQ) mass spectrometry (MS). SEC is a gentle protein separation technique, provided that the stationary and mobile phases are carefully selected. Using an optimized set of analytical conditions, it is possible to maintain the integrity of metalloprotein complexes (Persson et al., 2009; Husted et al., 2011). We systematically evaluate the essential and important factors required to obtain optimal chromatographic resolution while maintaining PSII integrity, focusing on choice of mobile phase, detergents, stabilizers, and the most suitable chromatographic columns for efficient protein fractionation and elution. The optimized method, with its multielement ability, enables the study of metal binding in PSII-LHCII super- and subcomplexes. To show the applicability of the method, we studied the metal profiles of barley thylakoids that had been isolated from plants with different levels of Mn deficiency. Mn binding in size-fractionated PSII complexes was evaluated in response to increasing Mn deficiency, and two genotypes differing in their tolerance to Mn deficiency were compared.     

Middle Ordovician Aporthophyla brachiopod fauna from the roof of the World,southern Tibet

A Darriwilian (late Middle Ordovician) brachiopod fauna from the Lower Formation of the Chiatsun Group at Jiacun, northern Nyalam, southern Tibet, consists of ten brachiopod species, forming a distinct Aporthophyla–Paralenorthis Association. Its taxonomic composition is typical of the Aporthophyla Fauna that occupied lower BA2 to upper BA3 benthic environments on sandy lime mud substrates. The occurrence of Paralenorthis in southern Tibet is confirmed for the first time, represented by P. costata sp. nov. Numerical analyses (PCA and CA) of 18 Darriwilian brachiopod faunas from ten palaeoplates or terranes indicate that: (1) the Aporthophyla Fauna was confined to a specific latitudinal belt although it had a wide lateral distribution from the large palaeocontinents of Gondwana to Laurentia; (2) the Saucrorthis Fauna, a typical late Middle Ordovician regional fauna, is limited to a much smaller area, marginal to the Gondwana supercontinent; (3) the strong provincialism persistent in the late Middle Ordovician contributed to increased gamma biodiversity during the Great Ordovician Biodiversification Event.     

Assessment of time patterns of activity and rest in full-shift recordings of trapezius muscle activity – Effects of the data processing procedure

The purpose of this paper was to compare the effects of different data reduction procedures on the values of variables characterizing the time pattern of trapezius muscle activity during full work shifts. Surface electromyography (EMG) of the right and left upper trapezius muscles were obtained from 40 young subjects in different occupations, mainly electricians, hairdressers and students. The target EMG variables were gap frequency, muscle rest, and the number and duration of episodes with sustained muscle activity (from 0.13 s to 30 min as minimum duration). These variables were derived from the EMG recordings using different Root Mean Square (RMS) windows (from 0.13 to 6.38 s), and discrimination levels between “activity” and “rest” (0.5%, 1% and 2% of maximal EMG).The results give basis for practical suggestions for EMG analyses of full work shifts. For most variables, a discrimination level of 0.5% EMG_max showed to be preferable. The time proportion of muscle rest and sustained muscle activity should, in general, be preferred over the corresponding frequency measures. Sustained muscle activity should be calculated using a RMS window between 1 and 3 s, and preferably be stated in terms of variables describing time proportions of activity. Uninterrupted activity episodes longer than 10 min proved not to be a useful variable due to limited occurrence in many work shifts.     

Reduction of nitrate in clayey subsoils controlled by geochemical and microbial barriers

Vertical distribution of redox zones, concentrations of redox‐sensitive constituents, numbers of aerobic heterotrophic bacteria, and potential denitrification activity were studied in 1‐m cores taken at the transition between the oxidized and reduced layers in two Danish clayey subsoils. Based on the matrix soil colors, a redox sequence of oxidized, suboxic, and reduced zones was identified at both sites. The geochemical composition of the oxidized brown colored zone (to depths of 2.6 and 3.2 m) was characterized by high concentrations of NO₃ ^? and low amounts of total organic carbon, exchangeable forms of NH₄ ⁺, Fe²⁺, and Mn²⁺, and structural Fe(II) in the clay minerals. In the underlying 20‐ to 30‐cm‐deep suboxic zone, decreasing NO₃ ^? concentrations were observed together with increasing amount of exchangeable forms of Fe²⁺ and Mn²⁺, and structural Fe(II). Finally, in the reduced grey zone, NO₃ ^? was no longer present and maximum concentrations of other redox sensitive constituents occurred. Throughout the subsoils, the distribution of exchangeable Fe²⁺ corresponded most closely to changes in the colors of redox zones. The low‐organic Havrebjerg site displayed geochemical profiles indicating that NO₃ ^? was chemically reduced by structural Fe(II) in the clay minerals of the suboxic zone, and that the Fe(II) formed a geochemical barrier for the downward progression of NO₃ ^?. Aerobic heterotrophic bacteria occurred only in low numbers at this site and potential denitrification activity was very low. In contrast, the Sparresholm site had a significant population of bacteria in the suboxic zone, which also contained a heterogeneous distribution of potential denitrification activity. Specific microsites with facilitated transport of soluble organic substrates are proposed to support the denitrification activity in a heterogeneous distribution, constituting a microbial barrier for downward progression of NO₃ ^? in this subsoil.     

Normalizing upper trapezius EMG amplitude: Comparison of different procedures

Different procedures have been used for normalization of upper trapezius electromyographic (EMG) amplitudes. This complicates comparisons between studies. The present study aimed at investigating the influence of some commonly used trapezius EMG normalization procedures on the results of ergonomic analyses, as well as the test-retest repeatability of these procedures. EMG activity from the upper trapezius was recorded during an occupational task. The EMG activity was then normalized by seven different normalization procedures. It was shown that at the group level, a unilateral shoulder elevation maximal voluntary electrical (MVE) activation procedure gave 1.2 times higher occupational load estimates than a corresponding bilateral MVE. At the group level, the median load during the occupational task was 1.6 times higher when expressed as %MVC (maximal voluntary contraction) obtained from a power regression of relative force on EMG amplitude than when expressed as %MVE determined from a single maximal shoulder elevation. Normalizations in terms of a submaximal reference voluntary electrical (RVE) activation had similar test-retest repeatability in terms of the coefficient of variation (CV: 11–13%) as normalizations in terms of an MVE (CV: 11–15%), but the power regression procedures had considerably larger CVs (21–36%). The paper provides a basis for comparing previous studies using different normalization methods, as well as a qualitative evaluation of normalization methods for future use.     

Can Cognitive Activities during Breaks in Repetitive Manual Work Accelerate Recovery from Fatigue? A Controlled Experiment

Neurophysiologic theory and some empirical evidence suggest that fatigue caused by physical work may be more effectively recovered during “diverting” periods of cognitive activity than during passive rest; a phenomenon of great interest in working life. We investigated the extent to which development and recovery of fatigue during repeated bouts of an occupationally relevant reaching task was influenced by the difficulty of a cognitive activity between these bouts. Eighteen male volunteers performed three experimental sessions, consisting of six 7-min bouts of reaching alternating with 3 minutes of a memory test differing in difficulty between sessions. Throughout each session, recordings were made of upper trapezius muscle activity using electromyography (EMG), heart rate and heart rate variability (HRV) using electrocardiography, arterial blood pressure, and perceived fatigue (Borg CR10 scale and SOFI). A test battery before, immediately after and 1 hour after the work period included measurements of maximal shoulder elevation strength (MVC), pressure pain threshold (PPT) over the trapezius muscles, and a submaximal isometric contraction. As expected, perceived fatigue and EMG amplitude increased during the physical work bouts. Recovery did occur between the bouts, but fatigue accumulated throughout the work period. Neither EMG changes nor recovery of perceived fatigue during breaks were influenced by cognitive task difficulty, while heart rate and HRV recovered the most during breaks with the most difficult task. Recovery of perceived fatigue after the 1 hour work period was also most pronounced for the most difficult cognitive condition, while MVC and PPT showed ambiguous patterns, and EMG recovered similarly after all three cognitive protocols. Thus, we could confirm that cognitive tasks between bouts of fatiguing physical work can, indeed, accelerate recovery of some factors associated with fatigue, even if benefits may be moderate and some responses may be equivocal. Our results encourage further research into combinations of physical and mental tasks in an occupational context.