Effect of Exercise on Mental Health and Health-related Quality of Life in Adults With Spinal Cord Injury: A Systematic Review and Meta-Analysis.

OBJECTIVES: To determine the effect of exercise interventions on mental health and health-related quality of life (HRQoL) in individuals with SCI. DATA SOURCES: We searched Embase, CINAHL, Medline, PsychINFO, and SPORTDiscus from inception to September 2023. STUDY SELECTION: We included randomized controlled trials that (1) involved participants ≥18 years old with a SCI; (2) administered an exercise intervention; and (3) measured subjective well-being, psychological well-being, social well-being, and/or HRQoL as outcomes. We reported standardized means differences (d) with a 95% confidence interval (CI), assessed the risk of bias by using the Revised Cochrane Risk-of-bias Tool for Randomized Trials (RoB 2), and the certainty of the evidence using GRADE. DATA SYNTHESIS: Nineteen studies (797 participants, mean age <65 years in every study) were included. Exercise improved overall well-being (d=0.494; 95% CI 0.268, 0.720; low certainty evidence), subjective well-being (d=0.543; 95% CI 0.270, 0.816; low certainty evidence), psychological well-being (d=0.499; 95% CI 0.193, 0.805; low certainty evidence), social well-being (d=0.452; 95% CI 0.151, 0.752; low certainty evidence), and HRQoL (d=0.323; 95% CI 0.072, 0.574; low certainty evidence). Four serious adverse events probably attributable to the interventions were reported in 3 studies. CONCLUSIONS: Exercise interventions can improve well-being and HRQoL in adults with SCI <65 years of age. Additional research is needed to determine effectiveness in adults ≥65 years of age.

The Effect of Sitting Duration on Peripheral Blood Pressure Responses to Prolonged Sitting, With and Without Interruption: A Systematic Review and Meta-Analysis.

BACKGROUND: A previous meta-analysis reported that: (i) an acute bout of prolonged uninterrupted sitting induces a significant increase in peripheral blood pressure (BP) and (ii) the increase in BP can be offset by interrupting the sitting bout with light aerobic activities such as walking. However, the temporal association between prolonged uninterrupted sitting and BP was not determined. A better understanding of temporality, for example, how long it takes BP to increase, will assist in prescribing sitting interruption strategies. OBJECTIVES: We aimed to determine: (1) the temporal association between the duration of uninterrupted sitting and BP and (2) whether regular sitting interruptions moderate the association between uninterrupted sitting and BP. DATA SOURCES: Electronic databases (PubMed, Web of Science, SPORTDiscus) were searched from inception to July 2022. Reference lists of eligible studies and relevant reviews were also screened. STUDY SELECTION: Inclusion criteria for objective (1) were: (i) participants aged ≥ 18 years; (ii) a prolonged sitting bout ≥ 1 h; and (iii) peripheral BP measurements (systolic BP, diastolic BP, and/or mean arterial pressure) at more than two timepoints during the sitting bout. Additional criteria for objective (2) were: (i) the sitting interruption strategy was implemented during the sitting bout (i.e., not prior to engaging in sitting) and (ii) the study included a control (uninterrupted sitting) condition or group. APPRAISAL AND SYNTHESIS METHODS: There were 1555 articles identified, of which 33 met inclusion criteria for objective (1). Of those articles, 20 met inclusion criteria for objective (2). To investigate the effect of sitting duration on the BP response, unstandardized b coefficients (mmHg/h) and 95% confidence intervals (CIs) were calculated using a three-level mixed-effect meta-regression. RESULTS: Increased sitting duration was positively associated with systolic BP (b = 0.42 mmHg/h, 95% CI 0.18-0.60), diastolic BP (b = 0.24 mmHg/h, 95% CI 0.06-0.42), and mean arterial pressure (b = 0.66 mmHg/h, 95% CI 0.36-0.90). In trials where sitting was interrupted, there was a significant decrease in systolic BP (b = - 0.24 mmHg/h, 95% CI - 0.42 to - 0.06) and diastolic BP (b = - 0.24 mmHg/h, 95% CI - 0.42 to - 0.12), and a non-significant change in mean arterial pressure (p = 0.69). CONCLUSIONS: Increased uninterrupted sitting duration results in greater increases in BP; however, regularly interrupting sitting may offset negative effects.

Efficient and Targeted siRNA Delivery to M2 Macrophages by Smart Polymer Blends for M1 Macrophage Repolarization as a Promising Strategy for Future Cancer Treatment.

Cancer remains an issue on a global scale. It is estimated that nearly 10 million people succumbed to cancer worldwide in 2020. New treatment options are urgently needed. A promising approach is a conversion of tumor-promoting M2 tumor-associated macrophages (TAMs) as part of the tumor microenvironment to tumor-suppressive M1 TAMs by small interfering RNA (siRNA). In this work, we present a well-characterized polymeric nanocarrier system capable of targeting M2 TAMs by a ligand-receptor interaction. Therefore, we developed a blended PEI-based polymeric nanoparticle system conjugated with mannose, which is internalized after interaction with macrophage mannose receptors (MMRs), showing low cytotoxicity and negligible IL-6 activation. The PEI-PCL-PEI (5 kDa-5 kDa-5 kDa) and Man-PEG-PCL (2 kDa-2 kDa) blended siRNA delivery system was optimized for maximum targeting capability and efficient endosomal escape by evaluation of different polymer and N/P ratios. The nanoparticles were formulated by surface acoustic wave-assisted microfluidics, achieving a size of ∼80 nm and a zeta potential of approximately +10 mV. Special attention was given to the endosomal escape as the so-called bottleneck of RNA drug delivery. To estimate the endosomal escape capability of the nanocarrier system, we developed a prediction method by evaluating the particle stability via the inflection temperature. Our predictions were then verified in an in vitro setting by applying confocal microscopy. For cellular experiments, however, human THP-1 cells were polarized to M2 macrophages by cytokine treatment and validated through MMR expression. To show the efficiency of the nanoparticle system, GAPDH and IκBα knockdown was performed in the presence or absence of an MMR blocking excess of mannan. Cellular uptake, GAPDH knockdown, and NF-κB western blot confirmed efficient mannose targeting. Herein, we presented a well-characterized nanoparticle delivery system and a promising approach for targeting M2 macrophages by a mannose-MMR interaction.

Chlorophyll biosynthesis under the control of arginine metabolism.

In natural environments, photosynthetic organisms adjust their metabolism to cope with the fluctuating availability of combined nitrogen sources, a growth-limiting factor. For acclimation, the dynamic degradation/synthesis of tetrapyrrolic pigments, as well as of the amino acid arginine, is pivotal; however, there has been no evidence that these processes could be functionally coupled. Using co-immunopurification and spectral shift assays, we found that in the cyanobacterium Synechocystis sp. PCC 6803, the arginine metabolism-related ArgD and CphB enzymes form protein complexes with Gun4, an essential protein for chlorophyll biosynthesis. Gun4 binds ArgD with high affinity, and the Gun4-ArgD complex accumulates in cells supplemented with ornithine, a key intermediate of the arginine pathway. Elevated ornithine levels restricted de novo synthesis of tetrapyrroles, which arrested the recovery from nitrogen deficiency. Our data reveal a direct crosstalk between tetrapyrrole biosynthesis and arginine metabolism that highlights the importance of balancing photosynthetic pigment synthesis with nitrogen homeostasis.

Assessing Empiric Antimicrobial Therapy With the Modified Dundee Classification for Nonpurulent Skin and Soft Tissue Infections in a Community Hospital System.

Background: The modified Dundee classification has recently been validated in various studies for nonpurulent skin and soft tissue infections. This has yet to be applied in the United States and within community hospital settings to optimize antimicrobial stewardship and ultimately patient care. Methods: A retrospective, descriptive analysis was performed of 120 adult patients admitted to St. Joseph's/Candler Health System for nonpurulent skin and soft tissue infections between January 2020 and September 2021. Patients were classified into their modified Dundee classes, and frequencies of concordance of their empiric antimicrobial regimens with this classification scheme in the emergency department and inpatient settings were compared, along with possible effect modifiers and possible exploratory measures associated with concordance. Results: Concordance with the modified Dundee classification for the emergency department and inpatient regimens was 10% and 15%, respectively, with broad-spectrum antibiotic use and concordance positively associated with illness severity. Due to substantial broad-spectrum antibiotic use, possible effect modifiers associated with concordance were unable to be validated, and overall no statistically significant differences among exploratory analyses across classification status were observed. Conclusions: The modified Dundee classification can help identify gaps in antimicrobial stewardship and excessive broad-spectrum antimicrobial usage toward optimizing patient care.

Multivalent binding kinetics resolved by fluorescence proximity sensing.

Multivalent protein interactors are an attractive modality for probing protein function and exploring novel pharmaceutical strategies. The throughput and precision of state-of-the-art methodologies and workflows for the effective development of multivalent binders is currently limited by surface immobilization, fluorescent labelling and sample consumption. Using the gephyrin protein, the master regulator of the inhibitory synapse, as benchmark, we exemplify the application of Fluorescence proximity sensing (FPS) for the systematic kinetic and thermodynamic optimization of multivalent peptide architectures. High throughput synthesis of +100 peptides with varying combinatorial dimeric, tetrameric, and octameric architectures combined with direct FPS measurements resolved on-rates, off-rates, and dissociation constants with high accuracy and low sample consumption compared to three complementary technologies. The dataset and its machine learning-based analysis deciphered the relationship of specific architectural features and binding kinetics and thereby identified binders with unprecedented protein inhibition capacity; thus, highlighting the value of FPS for the rational engineering of multivalent inhibitors.

Spray drying siRNA-lipid nanoparticles for dry powder pulmonary delivery.

While all the siRNA drugs on the market target the liver, the lungs offer a variety of currently undruggable targets which could potentially be treated with RNA therapeutics. Hence, local, pulmonary delivery of RNA nanoparticles could finally enable delivery beyond the liver. The administration of RNA drugs via dry powder inhalers offers many advantages related to physical, chemical and microbial stability of RNA and nanosuspensions. The present study was therefore designed to test the feasibility of engineering spray dried lipid nanoparticle (LNP) powders. Spray drying was performed using 5% lactose solution (m/V), and the targets were set to obtain nanoparticle sizes after redispersion of spray-dried powders around 150 nm, a residual moisture level below 5%, and RNA loss below 15% at maintained RNA bioactivity. The LNPs consisted of an ionizable cationic lipid which is a sulfur-containing analog of DLin-MC3-DMA, a helper lipid, cholesterol, and PEG-DMG encapsulating siRNA. Prior to the spray drying, the latter process was simulated with a novel dual emission fluorescence spectroscopy method to preselect the highest possible drying temperature and excipient solution maintaining LNP integrity and stability. Through characterization of physicochemical and aerodynamic properties of the spray dried powders, administration criteria for delivery to the lower respiratory tract were fulfilled. Spray dried LNPs penetrated the lung mucus layer and maintained bioactivity for >90% protein downregulation with a confirmed safety profile in a lung adenocarcinoma cell line. Additionally, the spray dried LNPs successfully achieved up to 50% gene silencing of the house keeping gene GAPDH in ex vivo human precision-cut lung slices at without increasing cytokine levels. This study verifies the successful spray drying procedure of LNP-siRNA systems maintaining their integrity and mediating strong gene silencing efficiency on mRNA and protein levels both in vitro and ex vivo. The successful spray drying procedure of LNP-siRNA formulations in 5% lactose solution creates a novel siRNA-based therapy option to target respiratory diseases such as lung cancer, asthma, COPD, cystic fibrosis and viral infections.

Physical activity and sedentary behavior in people with spinal cord injury: Mitigation strategies during COVID-19 on behalf of ACSM-EIM and HL-PIVOT.

People with spinal cord injury (SCI) face unique challenges during the COVID-19 pandemic, including greater risk of poor COVID-19-related outcomes, increased social isolation, and restricted access to important services. Furthermore, COVID-19 related restrictions have decreased already low levels of physical activity (PA) in this population. Therefore, the purpose of this commentary is to: 1) address the impact of COVID-19 on PA and sedentary behavior (SB) in people with SCI; 2) provide potential SB reduction strategies to guide future research; and 3) provide recommendations to increase PA and reduce SB on behalf of the American College of Sports Medicine Exercise is Medicine (ACSM-EIM) and Healthy Living for Pandemic Event Protection (HL-PIVOT) using a social-ecological model targeting the individual-, social environment-, physical environment-, and policy-level determinants of behavior in people with SCI.

Transporter characterisation reveals aminoethylphosphonate mineralisation as a key step in the marine phosphorus redox cycle.

The planktonic synthesis of reduced organophosphorus molecules, such as alkylphosphonates and aminophosphonates, represents one half of a vast global oceanic phosphorus redox cycle. Whilst alkylphosphonates tend to accumulate in recalcitrant dissolved organic matter, aminophosphonates do not. Here, we identify three bacterial 2-aminoethylphosphonate (2AEP) transporters, named AepXVW, AepP and AepSTU, whose synthesis is independent of phosphate concentrations (phosphate-insensitive). AepXVW is found in diverse marine heterotrophs and is ubiquitously distributed in mesopelagic and epipelagic waters. Unlike the archetypal phosphonate binding protein, PhnD, AepX has high affinity and high specificity for 2AEP (Stappia stellulata AepX Kd 23 ± 4 nM; methylphosphonate Kd 3.4 ± 0.3 mM). In the global ocean, aepX is heavily transcribed (~100-fold>phnD) independently of phosphate and nitrogen concentrations. Collectively, our data identifies a mechanism responsible for a major oxidation process in the marine phosphorus redox cycle and suggests 2AEP may be an important source of regenerated phosphate and ammonium, which are required for oceanic primary production.

How the O2-dependent Mg-protoporphyrin monomethyl ester cyclase forms the fifth ring of chlorophylls.

Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase catalyses the formation of the isocyclic ring, producing protochlorophyllide a and contributing substantially to the absorption properties of chlorophylls and bacteriochlorophylls. The O2-dependent cyclase is found in both oxygenic phototrophs and some purple bacteria. We overproduced the simplest form of the cyclase, AcsF, from Rubrivivax gelatinosus, in Escherichia coli. In biochemical assays the di-iron cluster within AcsF is reduced by ferredoxin furnished by NADPH and ferredoxin:NADP+ reductase, or by direct coupling to Photosystem I photochemistry, linking cyclase to the photosynthetic electron transport chain. Kinetic analyses yielded a turnover number of 0.9 min-1, a Michaelis-Menten constant of 7.0 µM for MgPME and a dissociation constant for MgPME of 0.16 µM. Mass spectrometry identified 131-hydroxy-MgPME and 131-keto-MgPME as cyclase reaction intermediates, revealing the steps that form the isocyclic ring and completing the work originated by Sam Granick in 1950.

The active site of magnesium chelatase.

The insertion of magnesium into protoporphyrin initiates the biosynthesis of chlorophyll, the pigment that underpins photosynthesis. This reaction, catalysed by the magnesium chelatase complex, couples ATP hydrolysis by a ChlID motor complex to chelation within the ChlH subunit. We probed the structure and catalytic function of ChlH using a combination of X-ray crystallography, computational modelling, mutagenesis and enzymology. Two linked domains of ChlH in an initially open conformation of ChlH bind protoporphyrin IX, and the rearrangement of several loops envelops this substrate, forming an active site cavity. This induced fit brings an essential glutamate (E660), proposed to be the key catalytic residue for magnesium insertion, into proximity with the porphyrin. A buried solvent channel adjacent to E660 connects the exterior bulk solvent to the active site, forming a possible conduit for the delivery of magnesium or abstraction of protons.

Quantifying the Interaction of Phosphite with ABC Transporters: MicroScale Thermophoresis and a Novel His-Tag Labeling Approach.

The combination of MicroScale Thermophoresis (MST) and near-native site-specific His-tag labeling enables simple, robust, and reliable determination of the binding affinity between proteins and ligands. To demonstrate its applicability for periplasmic proteins, we provide a detailed protocol for determination of the binding affinity of phosphite to three ABC transporter periplasmic-binding proteins from environmental microorganisms. ABC transporters are central to many important biomedical phenomena, including resistance of cancers and pathogenic microbes to drugs. The protocol described here can be used to quantify protein-ligand and protein-protein interactions for other soluble, membrane-associated and integral membrane proteins.

Phosphite binding by the HtxB periplasmic binding protein depends on the protonation state of the ligand.

Phosphorus acquisition is critical for life. In low phosphate conditions, some species of bacteria have evolved mechanisms to import reduced phosphorus compounds, such as phosphite and hypophosphite, as alternative phosphorus sources. Uptake is facilitated by high-affinity periplasmic binding proteins (PBPs) that bind cargo in the periplasm and shuttle it to an ATP-binding cassette (ABC)-transporter in the bacterial inner membrane. PtxB and HtxB are the PBPs responsible for binding phosphite and hypophosphite, respectively. They recognize the P-H bond of phosphite/hypophosphite via a conserved P-H...π interaction, which confers nanomolar dissociation constants for their respective ligands. PtxB also has a low-level binding affinity for phosphate and hypophosphite, whilst HtxB can facilitate phosphite uptake in vivo. However, HtxB does not bind phosphate, thus the HtxBCDE transporter has recently been successfully exploited for biocontainment of genetically modified organisms by phosphite-dependent growth. Here we use a combination of X-ray crystallography, NMR and Microscale Thermophoresis to show that phosphite binding to HtxB depends on the protonation state of the ligand, suggesting that pH may effect the efficiency of phosphite uptake by HtxB in biotechnology applications.

The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase.

Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP2--dependent insertion of a Mg2+ ion into protoporphyrin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species, respectively). The D and I subunits are members of the AAA+ (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro, ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH complex. Quantitative analysis using microscale thermophoresis showed magnesium-dependent binding (Kd 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD-H interaction was investigated using chemical cross-linking coupled with mass spectrometry (XL-MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. The interaction site between the AAA+ motor and the chelatase domain of magnesium chelatase will be essential for understanding how free energy from the hydrolysis of ATP on the AAA+ ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH.

Probing the quality control mechanism of the Escherichia coli twin-arginine translocase with folding variants of a de novo-designed heme protein.

Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin-arginine translocase (Tat). The Tat machinery exports folded and cofactor-containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality-control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one, or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system's quality-control mechanism.

The molecular basis of phosphite and hypophosphite recognition by ABC-transporters.

Inorganic phosphate is the major bioavailable form of the essential nutrient phosphorus. However, the concentration of phosphate in most natural habitats is low enough to limit microbial growth. Under phosphate-depleted conditions some bacteria utilise phosphite and hypophosphite as alternative sources of phosphorus, but the molecular basis of reduced phosphorus acquisition from the environment is not fully understood. Here, we present crystal structures and ligand binding affinities of periplasmic binding proteins from bacterial phosphite and hypophosphite ATP-binding cassette transporters. We reveal that phosphite and hypophosphite specificity results from a combination of steric selection and the presence of a P-H…π interaction between the ligand and a conserved aromatic residue in the ligand-binding pocket. The characterisation of high affinity and specific transporters has implications for the marine phosphorus redox cycle, and might aid the use of phosphite as an alternative phosphorus source in biotechnological, industrial and agricultural applications.

Nanomechanical and Thermophoretic Analyses of the Nucleotide-Dependent Interactions between the AAA(+) Subunits of Magnesium Chelatase.

In chlorophyll biosynthesis, the magnesium chelatase enzyme complex catalyzes the insertion of a Mg(2+) ion into protoporphyrin IX. Prior to this event, two of the three subunits, the AAA(+) proteins ChlI and ChlD, form a ChlID-MgATP complex. We used microscale thermophoresis to directly determine dissociation constants for the I-D subunits from Synechocystis, and to show that the formation of a ChlID-MgADP complex, mediated by the arginine finger and the sensor II domain on ChlD, is necessary for the assembly of the catalytically active ChlHID-MgATP complex. The N-terminal AAA(+) domain of ChlD is essential for complex formation, but some stability is preserved in the absence of the C-terminal integrin domain of ChlD, particularly if the intervening polyproline linker region is retained. Single molecule force spectroscopy (SMFS) was used to determine the factors that stabilize formation of the ChlID-MgADP complex at the single molecule level; ChlD was attached to an atomic force microscope (AFM) probe in two different orientations, and the ChlI subunits were tethered to a silica surface; the probability of subunits interacting more than doubled in the presence of MgADP, and we show that the N-terminal AAA(+) domain of ChlD mediates this process, in agreement with the microscale thermophoresis data. Analysis of the unbinding data revealed a most probable interaction force of around 109 pN for formation of single ChlID-MgADP complexes. These experiments provide a quantitative basis for understanding the assembly and function of the Mg chelatase complex.

The catalytic power of magnesium chelatase: a benchmark for the AAA(+) ATPases.

In the first committed reaction of chlorophyll biosynthesis, magnesium chelatase couples ATP hydrolysis to the thermodynamically unfavorable Mg(2+) insertion into protoporphyrin IX (ΔG°' of circa 25-33 kJ·mol(-1) ). We explored the thermodynamic constraints on magnesium chelatase and demonstrate the effect of nucleotide hydrolysis on both the reaction kinetics and thermodynamics. The enzyme produces a significant rate enhancement (kcat /kuncat of 400 × 10(6) m) and a catalytic rate enhancement, kcat/KmDIXK0.5Mgkuncat, of 30 × 10(15) m(-1) , increasing to 300 × 10(15) m(-1) with the activator protein Gun4. This is the first demonstration of the thermodynamic benefit of ATP hydrolysis in the AAA(+) family.

Five glutamic acid residues in the C-terminal domain of the ChlD subunit play a major role in conferring Mg(2+) cooperativity upon magnesium chelatase.

Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis by inserting a Mg(2+) ion into protoporphyrin IX in an ATP-dependent manner. The cyanobacterial (Synechocystis) and higher-plant chelatases exhibit a complex cooperative response to free magnesium, while the chelatases from Thermosynechococcus elongatus and photosynthetic bacteria do not. To investigate the basis for this cooperativity, we constructed a series of chimeric ChlD proteins using N-terminal, central, and C-terminal domains from Synechocystis and Thermosynechococcus. We show that five glutamic acid residues in the C-terminal domain play a major role in this process.