Application of a theorem by bright to the generalized tracer system

Bright (1973) has proved a theorem for a specific class of compartment systems. The theorem is here generalized, and it suggests that, occasionally, the steady-state whole-body mass of certain substances can be estimated from measurements of the mass concentration (e.g. specific activity) in blood alone, i.e. without cumbersome recordings of whole-body retention curves.     

Longitudinal deep sequencing informs vector selection and future deployment strategies for transmissible vaccines

Vaccination is a powerful tool in combating infectious diseases of humans and companion animals. In most wildlife, including reservoirs of emerging human diseases, achieving sufficient vaccine coverage to mitigate disease burdens remains logistically unattainable. Virally vectored “transmissible” vaccines that deliberately spread among hosts are a potentially transformative, but still theoretical, solution to the challenge of immunising inaccessible wildlife. Progress towards real-world application is frustrated by the absence of frameworks to guide vector selection and vaccine deployment prior to major in vitro and in vivo investments in vaccine engineering and testing. Here, we performed deep sequencing on field-collected samples of Desmodus rotundus betaherpesvirus (DrBHV), a candidate vector for a transmissible vaccine targeting vampire bat–transmitted rabies. We discovered 11 strains of DrBHV that varied in prevalence and geographic distribution across Peru. The phylogeographic structure of DrBHV strains was predictable from both host genetics and landscape topology, informing long-term DrBHV-vectored vaccine deployment strategies and identifying geographic areas for field trials where vaccine spread would be naturally contained. Multistrain infections were observed in 79% of infected bats. Resampling of marked individuals over 4 years showed within-host persistence kinetics characteristic of latency and reactivation, properties that might boost individual immunity and lead to sporadic vaccine transmission over the lifetime of the host. Further, strain acquisitions by already infected individuals implied that preexisting immunity and strain competition are unlikely to inhibit vaccine spread. Our results support the development of a transmissible vaccine targeting a major source of human and animal rabies in Latin America and show how genomics can enlighten vector selection and deployment strategies for transmissible vaccines.

Deep sequencing of vampire bat betaherpesviruses reveals prevalent multi-strain, multi-year infections with predictable patterns of spread and no evidence of cross immunity, supporting the herpesvirus as a promising transmissible vaccine candidate for bat rabies.     

Extracorporeal circulation in the isolated in situ lamb placenta: hemodynamic characteristics.

Decreased placental perfusion and respiratory gas exchange have been observed after experimental fetal cardiopulmonary bypass (CPB). To better characterize placental hemodynamics during CPB, seven isolated in situ lamb placentas were placed on a CPB circuit by use of umbilical arterial and venous cannulation. Measures were taken to simulate normal placental hemodynamics. Perfusion flow rates were varied from 15 to 300 ml.min-1.kg fetal wt-1 during normothermia and hypothermia. Placental vascular resistance (PVR) remained constant when perfusion pressure and flow were varied above 40 mmHg and 150 ml.min-1.kg-1, respectively. Below these values, PVR varied inversely. This increase in PVR was more marked when CPB was performed with hypothermia than with normothermia. The clinical implication is that decreased placental flow and pressure on CPB may lead to a vicious cycle, resulting in further impairment of placental perfusion and respiratory gas exchange. Hypothermia promotes this impairment.     

Dystrophic Spinal Deformities in a Neurofibromatosis Type 1 Murine Model

Despite the high prevalence and significant morbidity of spinal anomalies in neurofibromatosis type 1 (NF1), the pathogenesis of these defects remains largely unknown. Here, we present two murine models: Nf1^flox/−;PeriCre and Nf1^flox/−;Col.2.3Cre mice, which recapitulate spinal deformities seen in the human disease. Dynamic histomorphometry and microtomographic studies show recalcitrant bone remodeling and distorted bone microarchitecture within the vertebral spine of Nf1^flox/−;PeriCre and Nf1^flox/−;Col2.3Cre mice, with analogous histological features present in a human patient with dystrophic scoliosis. Intriguingly, 36–60% of Nf1^flox/−;PeriCre and Nf1^flox/−;Col2.3Cre mice exhibit segmental vertebral fusion anomalies with boney obliteration of the intervertebral disc (IVD). While analogous findings have not yet been reported in the NF1 patient population, we herein present two case reports of IVD defects and interarticular vertebral fusion in patients with NF1. Collectively, these data provide novel insights regarding the pathophysiology of dystrophic spinal anomalies in NF1, and provide impetus for future radiographic analyses of larger patient cohorts to determine whether IVD and vertebral fusion defects may have been previously overlooked or underreported in the NF1 patient population.     

STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions,and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes

In plants and algae, the serine/threonine kinase STN7/STT7, orthologous protein kinases in Chlamydomonas reinhardtii and Arabidopsis (Arabidopsis thaliana), respectively, is an important regulator in acclimation to changing light environments. In this work, we assessed STT7-dependent protein phosphorylation under high light in C. reinhardtii, known to fully induce the expression of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3) and a nonphotochemical quenching mechanism, in relationship to anoxia where the activity of cyclic electron flow is stimulated. Our quantitative proteomics data revealed numerous unique STT7 protein substrates and STT7-dependent protein phosphorylation variations that were reliant on the environmental condition. These results indicate that STT7-dependent phosphorylation is modulated by the environment and point to an intricate chloroplast phosphorylation network responding in a highly sensitive and dynamic manner to environmental cues and alterations in kinase function. Functionally, the absence of the STT7 kinase triggered changes in protein expression and photoinhibition of photosystem I (PSI) and resulted in the remodeling of photosynthetic complexes. This remodeling initiated a pronounced association of LHCSR3 with PSI-LIGHT HARVESTING COMPLEX I (LHCI)-ferredoxin-NADPH oxidoreductase supercomplexes. Lack of STT7 kinase strongly diminished PSII-LHCII supercomplexes, while PSII core complex phosphorylation and accumulation were significantly enhanced. In conclusion, our study provides strong evidence that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments and gives new insights into acclimation strategies to these environmental conditions.Oxygenic photosynthesis converts solar energy into chemical energy. This energy is utilized for carbon dioxide assimilation, allowing the formation of complex organic material. Plant photosynthesis is performed by a series of reactions in and at the thylakoid membrane, resulting in light-dependent water oxidation, NADP reduction, and ATP formation (Whatley et al., 1963). These light reactions are catalyzed by two photosystems (PSI and PSII). A third multiprotein complex, also embedded in the thylakoid membrane, is the cytochrome b₆f (cyt b₆f) complex that links photosynthetic electron transfer processes between the two photosystems and functions in proton translocation. The ATP synthase takes advantage of the proton-motive force that is generated by the light reactions (Mitchell, 1961) to produce ATP. ATP and NADPH, generated through linear electron flow from PSII to PSI, drive the Calvin-Benson-Bassham cycle (Bassham et al., 1950) to fix CO₂. Alternatively, cyclic electron flow (CEF) between PSI and the cyt b₆f complex solely produces ATP (Arnon, 1959).Under normal growth conditions, CEF provides additionally required ATP for CO₂ fixation (Lucker and Kramer, 2013), counteracts overreduction of the PSI acceptor side under stressful environmental cues, and readjusts the ATP poise, leading to increased lumen acidification important for photoprotection (Alric, 2010; Peltier et al., 2010; Leister and Shikanai, 2013; Shikanai, 2014). In microalgae and vascular plants, CEF relies on the NAD(P)H dehydrogenase-dependent and/or PROTON GRADIENT REGULATION5 (PGR5)-related pathways (Munekage et al., 2002, 2004; Petroutsos et al., 2009; Tolleter et al., 2011; Johnson et al., 2014). For both pathways, supercomplexes consisting of PSI-LIGHT HARVESTING COMPLEX I (LHCI) and components of the respective electron transfer routes have been identified. In Arabidopsis (Arabidopsis thaliana), a unique NAD(P)H dehydrogenase-PSI supercomplex with a molecular mass of more than 1,000 kD was discovered (Peng et al., 2008). From Chlamydomonas reinhardtii, Iwai et al. (2010) isolated a protein supercomplex composed of PSI-LHCI, LHCII, the cyt b₆/f complex, ferredoxin-NADPH oxidoreductase (FNR), and PROTON GRADIENT REGULATION-LIKE1 (PGRL1).PGRL1 and PGR5 interact physically in Arabidopsis and associate with PSI to allow the operation of CEF (DalCorso et al., 2008). Functional data suggest that PGRL1 might operate as a ferredoxin-plastoquinone reductase (Hertle et al., 2013). The PGRL1-containing CEF supercomplex isolated from C. reinhardtii is capable of CEF under in vitro conditions in the presence of exogenously added soluble plastocyanin and ferredoxin (Iwai et al., 2010). Terashima et al. (2012) isolated a CEF supercomplex of similar composition from anaerobic growth conditions that was active in vitro and contained proteins such as the chloroplast-localized Ca²⁺ sensor CAS and ANAEROBIC RESPONSE1 (ANR1), which were also shown to be functionally important for efficient CEF in the alga. Notably, it was suggested that the onset of CEF in C. reinhardtii is redox controlled (Takahashi et al., 2013).It has been demonstrated that efficient CEF is crucial for successful acclimation to excess light (Munekage et al., 2004; Dang et al., 2014; Johnson et al., 2014; Kukuczka et al., 2014). The most rapid response to excess light, however, relies on a mechanism called nonphotochemical quenching (NPQ). The fastest constituent of NPQ is energy-dependent (qE) quenching, which operates at a time scale of seconds to minutes and regulates the thermal dissipation of excess absorbed light energy, thereby providing effective photoprotection. In vascular plants, the PSII protein PSII SUBUNIT S is essential for qE (Li et al., 2000), whereas qE induction in the green alga C. reinhardtii is mediated by LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3), an ancient light-harvesting protein that is missing in vascular plants (Peers et al., 2009). CEF and qE are complementary for acclimation to excess light, as double mutants deficient in both mechanisms possess additive phenotypes and are highly sensitive to light (Kukuczka et al., 2014). Another constituent of NPQ is the quenching by state transitions. State transitions are important to balance the excitation energy between PSI and PSII (Bonaventura and Myers, 1969; Murata, 1969). Under light conditions where PSII is preferentially excited, both PSII core and LHCII proteins become phosphorylated (Lemeille and Rochaix, 2010). As a consequence, phosphorylated LHCII proteins detach from PSII and partly connect to PSI (state 2). Under conditions where PSI excitation is predominant, this process is reversed. LHCII proteins are dephosphorylated and associate with PSII (state 1). The extent of state transition between vascular plants such as Arabidopsis and C. reinhardtii differs significantly. The proportion of mobile LHCII antenna is about 80% in the alga, whereas in Arabidopsis, only 15% to 20% of LHCII is transferred to PSI under state 2 conditions (Lemeille and Rochaix, 2010). However, the large increase in PSI antenna size in C. reinhardtii has recently been challenged (Nagy et al., 2014; Ünlü et al., 2014): while 70% to 80% of mobile LHCII detached from PSII in response to transition to state 2 conditions, only a fraction of about 20% functionally attached to PSI.Phosphorylation of LHC proteins requires the function of the STT7 kinase or its ortholog STN7 in C. reinhardtii or Arabidopsis, respectively. In the absence of the STT7/STN7 kinase, the initiation of state transitions is blocked (Depège et al., 2003; Bellafiore et al., 2005). The mobile LHCII fraction of C. reinhardtii includes the two monomeric minor LHCII antenna proteins, CP26 and CP29 (encoded by lhcb5 and lhcb4 genes), and the major chlorophyll a/b binding protein of LHCII, LHCBM5 (Takahashi et al., 2006), but also the LHCSR3 protein was suggested to migrate during state transitions (Allorent et al., 2013). Takahashi et al. (2014) suggested that only CP29 and LHCBM5 directly associate with PSI to form the PSI-LHCI-LHCII supercomplex, while the binding of CP26 could occur indirectly or via the other two proteins. However, it is not yet known whether STT7 directly phosphorylates the LHCII proteins or if this takes place as part of a kinase cascade (Rochaix, 2007). Nevertheless, the direct interaction between STT7 and the LHCII proteins is quite likely, since none of the other chloroplast kinases was found to be specifically required for LHCII phosphorylation (Rochaix, 2014). The activity of the STT7 kinase is mainly determined by the redox status of the plastoquinone pool (Vener et al., 1997; Zito et al., 1999). The identification of a PROTEIN PHOSPHATASE 2C (PP2C)-type phosphatase responsible for the dephosphorylation of the LHCII proteins in Arabidopsis has been described by two studies in parallel pointing to the fact that this enzyme, called PROTEIN PHOSPHATASE1/THYLAKOID-ASSOCIATED PHOSPHATASE38, acts directly on phosphorylated LHCII proteins, in particular when they are associated with the PSI-LHCI supercomplex (Pribil et al., 2010; Shapiguzov et al., 2010). Moreover, it is not known whether these phosphatases are constitutively active or if they are regulated by other means, for example through the redox state of the plastoquinone pool. Nonetheless, both enzymes are conserved in land plants and exhibit orthologous proteins in C. reinhardtii (Rochaix et al., 2012).Another kinase related to STN7/STT7 is encoded in the Arabidopsis and C. reinhardtii genomes and named STN8 and STATE TRANSITION-LIKE1 (STL1), respectively. STN8 is involved in PSII core subunit phosphorylation and influences the repair of PSII after photodamage (Bonardi et al., 2005; Vainonen et al., 2005). Remarkably, the disassembly of the PSII holocomplex is inhibited in STN7/STN8 double mutants (Tikkanen et al., 2008; Fristedt et al., 2009; Dietzel et al., 2011; Nath et al., 2013), suggesting that the phosphorylation of core subunits is required for PSII disassembly. It was further suggested that STN8 controls the transition between linear electron flow and CEF by the phosphorylation of PGRL1 in Arabidopsis (Reiland et al., 2011). As described for STN7, the activity of STN8 is probably regulated via the redox state of the plastoquinone pool (Bennett, 1991; Fristedt et al., 2009). Notably, the action of STN8 is counteracted by a chloroplast PP2C phosphatase (Samol et al., 2012), allowing for the fast reversibility of STN8-mediated acclimation responses. Thus, it appears that an intricate regulatory network of chloroplast protein kinases and phosphatases evolved in vascular plants and algae that drives the acclimation response to various environmental cues, including excess and changing light settings (Rochaix et al., 2012). As STN7/STT7 and STN8/STL1 kinase activities appear to be controlled by the redox poise of the plastoquinone pool, the plastoquinone pool would be a central player in these acclimation responses. On the other hand, the kinases themselves are subjected to phosphorylation (Reiland et al., 2009, 2011; Lemeille et al., 2010; Wang et al., 2013). However, the functional consequences of this phosphorylation are unknown.Recent comparative analyses revealed the presence of at least 15 distinct chloroplast protein kinases, suggesting an intricate kinase phosphorylation network in the chloroplast (Bayer et al., 2012). Generally, the phosphorylation of proteins is one of the most abundant posttranslational modifications. In complex eukaryotic systems, protein phosphorylation occurs most frequently on Ser followed by Thr residues, whereas protein phosphorylation of Tyr residues (1,800:200:1) is comparatively rare (Hunter, 1998; Mann et al., 2002). Protein phosphorylation is a general phenomenon in vivo; it is assumed that about one-third of all proteins are phosphorylated at a given time (Cohen, 2000; Ahn and Resing, 2001; Venter et al., 2001; Manning et al., 2002; Knight et al., 2003). A recent large-scale quantitative evaluation of human proteomic data strengthened the importance of protein phosphorylation for cellular function and human biology (Wilhelm et al., 2014). The C. reinhardtii and Arabidopsis genomes encode large kinase families (Arabidopsis Genome Initiative, 2000; Kerk et al., 2002; Merchant et al., 2007), supporting the view that protein phosphorylation also plays an important role in a plant’s life cycle. It is thus evident that the understanding of protein phosphorylation, including the specificity of residues phosphorylated or dephosphorylated in response to cellular as well as environmental factors, is one key to understanding the complex functional biological networks at the whole-system level. Likewise, it is crucial to design experimental setups allowing the linkage between phosphorylation events and particular physiological consequences to be elucidated.In this regard, we designed experiments to investigate STT7 kinase-dependent phosphorylation dynamics in C. reinhardtii in response to high light and anoxia, employing quantitative proteomics in conjunction with in-depth physiological characterization. These conditions are particularly interesting, as high light conditions are known to fully induce LHCSR3 protein expression and qE, while anoxia promotes CEF activity. Recently, it was demonstrated that qE and CEF are complementary and crucial in acclimation to these environmental cues (Kukuczka et al., 2014). Notably, LHCSR3 phosphorylation was suggested to depend on STT7 function (Bonente et al., 2011), while CEF supercomplex formation was found to be independent of STT7 kinase function (Takahashi et al., 2013), indicating that STT7 function might impact the acclimation to high light and anoxia in different ways. However, our quantitative proteomics and physiological data reveal that STT7-dependent variations in protein phosphorylation profiles have similar dramatic phenotypic consequences in both conditions, strongly suggesting that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments.     

Random mutagenesis of the prokaryotic peptide transporter YdgR identifies potential periplasmic gating residues

The peptide transporter (PTR) family represents a group of proton-coupled secondary transporters responsible for bulk uptake of amino acids in the form of di- and tripeptides, an essential process employed across species ranging from bacteria to humans. To identify amino acids critical for peptide transport in a prokaryotic PTR member, we have screened a library of mutants of the Escherichia coli peptide transporter YdgR using a high-throughput substrate uptake assay. We have identified 35 single point mutations that result in a full or partial loss of transport activity. Additional analysis, including homology modeling based on the crystal structure of the Shewanella oneidensis peptide transporter PepT(so), identifies Glu(56) and Arg(305) as potential periplasmic gating residues. In addition to providing new insights into transport by members of the PTR family, these mutants provide valuable tools for further study of the mechanism of peptide transport.