Mn-porphyrins in a four-helix bundle participate in photo-induced electron transfer with a bacterial reaction center.

Hybrid complexes incorporating synthetic Mn-porphyrins into an artificial four-helix bundle domain of bacterial reaction centers created a system to investigate new electron transfer pathways. The reactions were initiated by illumination of the bacterial reaction centers, whose primary photochemistry involves electron transfer from the bacteriochlorophyll dimer through a series of electron acceptors to the quinone electron acceptors. Porphyrins with diphenyl, dimesityl, or fluorinated substituents were synthesized containing either Mn or Zn. Electrochemical measurements revealed potentials for Mn(III)/Mn(II) transitions that are ~ 0.4 V higher for the fluorinated Mn-porphyrins than the diphenyl and dimesityl Mn-porphyrins. The synthetic porphyrins were introduced into the proteins by binding to a four-helix bundle domain that was genetically fused to the reaction center. Light excitation of the bacteriochlorophyll dimer of the reaction center resulted in new derivative signals, in the 400 to 450 nm region of light-minus-dark spectra, that are consistent with oxidation of the fluorinated Mn(II) porphyrins and reduction of the diphenyl and dimesityl Mn(III) porphyrins. These features recovered in the dark and were not observed in the Zn(II) porphyrins. The amplitudes of the signals were dependent upon the oxidation/reduction midpoint potentials of the bacteriochlorophyll dimer. These results are interpreted as photo-induced charge-separation processes resulting in redox changes of the Mn-porphyrins, demonstrating the utility of the hybrid artificial reaction center system to establish design guidelines for novel electron transfer reactions.

Generation of lytic natural killer 1.1+, Ly-49- cells from multipotential murine bone marrow progenitors in a stroma-free culture: definition of cytokine requirements and developmental intermediates.

We have developed a stroma-free culture system in which mouse marrow or thymus cells, known to be enriched for lymphoid progenitors, can be driven to generate natural killer (NK) cells. Culture of lineage marker (Lin)-, c-kit+, Sca2+, interleukin (IL)-2/15Rbeta (CD122)- marrow cells in IL-6, IL-7, stem cell factor (SCF), and flt3 ligand (flt3-L) for 5-6 d followed by IL-15 alone for an additional 4-5 d expanded the starting population 30-40-fold and gave rise to a virtually pure population of NK1.1+, CD3- cells. Preculture in IL-6, IL-7, SCF, and flt3-L was necessary for inducing IL-15 responsiveness in the progenitors because the cells failed to significantly expand when cultured in IL-15 alone from the outset. Although culture of the sorted progenitors in IL-6, IL-7, SCF, and flt3-L for the entire 9-11-d culture period caused significant expansion, no lytic NK1.1+ cells were generated if IL-15 was not added, demonstrating a critical role for IL-15 in NK differentiation. Thus, two distinct populations of NK progenitors, IL-15 unresponsive and IL-15 responsive, have been defined. Similar results were obtained with Lin-, CD44+, CD25-, c-kit+ lymphoid progenitors obtained from adult thymus. The NK cells generated by this protocol lysed the NK-sensitive target YAC-1 and expressed markers of mature NK cells with the notable absence of Ly-49 major histocompatibility complex (MHC) receptors. However, despite the apparent lack of these inhibitory MHC receptors, the NK cells generated could distinguish MHC class I+ from class I- syngeneic targets, suggesting the existence of novel class I receptors.

Novel genetic regulation of T helper 1 (Th1)/Th2 cytokine production and encephalitogenicity in inbred mouse strains.

Development of T helper cell (Th)1 or Th2 cytokine responses is essential for effector and regulatory functions of T helper cells. We have compared cytokine profiles of myelin basic protein (MBP) Ac1-16 peptide-specific T helper cells from inbred mouse strains expressing identical k haplotype-derived MHC class II molecules B10.A and B10.BR, B10.BR T cell lines (TCL) produced Th1 cytokines (including high levels of TNF-alpha) and induced experimental autoimmune encephalomyelitis after adoptive transfer. In contrast, B10.A TCL produced Th2 cytokines (including low levels of TNF-alpha) and were poorly encephalitogenic. The contributions of the genetic origin of the T cells and the APC were explored. Serial restimulations of the B10.BR TCL with B10.A or (B10.A x B10.BR) F1 splenic antigen presenting cells (APC) during the establishment of TCL markedly reduced both Th1 cytokine production and encephalitogenicity. In addition, a single restimulation with B10. A splenic APC reduced IFN-gamma and TNF-alpha production by established Th1 MBP-specific Ak-restricted B10.BR TCL and by a Th1 KLH-specific, Ek-restricted B10.BR T cell clone. These studies suggest that B10.A and B10.BR APC differ in their ability to stimulate IFN-gamma and TNF-alpha production by mature Th1 cells and also influence their Th1/Th2 commitment in vivo. The nature of the downregulatory activity of B10.A APC on IFN-gamma and TNF-alpha production was explored. 2-hour supernatants from antigen-activated B10.A APC/TCL cultures or from B10.A APC activated by LPS had the same inhibitory effects on IFN-gamma and TNF-alpha production by B10.BR TCL. The downregulatory effects of B10.A APC are independent of TNF-alpha, IL-4, IL-10, IL-12p40, IFN-gamma, IL-13, TGF-beta, and PGE2. Thus, genetic difference(s) between B10.A and B10.BR APC appear(s) to control the production or activity of a novel soluble cytokine regulatory factor that influences Th1/Th2 commitment and controls production of IFN-gamma and TNF-alpha by mature Th1 cells.

Domain-wall depinning assisted by pure spin currents.

We study the depinning of domain walls by pure diffusive spin currents in a nonlocal spin valve structure based on two ferromagnetic Permalloy elements with copper as the nonmagnetic spin conduit. The injected spin current is absorbed by the second Permalloy structure with a domain wall, and from the dependence of the wall depinning field on the spin current density we find an efficiency of 6×10{-14} T/(A/m{2}), which is more than an order of magnitude larger than for conventional current induced domain-wall motion. Theoretically we find that this high efficiency arises from the surface torques exerted by the absorbed spin current that lead to efficient depinning.

Mimicking photosynthesis.

Although the concept of an artificial photosynthetic reaction center that mimics natural electron-and energy-transfer processes is an old one, in recent years major advances have occurred. In this review, some relatively simple molecular dyads that mimic certain aspects of photosynthetic electron transfer and singlet or triplet energy transfer are described. Dyads of this type have proven to be extremely useful for elucidating basic photochemical principles. In addition, their limitations, particularly in the area of temporal stabilization of electronic charge separation, have inspired the development of much more complex multicomponent molecular devices. The use of the basic principles of photoinitiated electron transfer to engineer desirable properties into the more complex species is exemplified. The multiple electrontransfer pathways available with these molecules make it possible to fine-tune the systems in ways that are impossible with simpler molecules. The study of these devices not only contributes to our understanding of natural photosynthesis, but also aids in the design of artificial solar energy harvesting systems and provides an entry into the nascent field of molecular electronics.

Direct Observation of CIO from Chlorine Nitrate Photolysis.

Chlorine nitrate photolysis has been investigated with the use of a molecular beam technique. Excitation at both 248 and 193 nanometers led to photodissociation by two pathways, CIONO(2) --> CIO + NO()2 and CIONO(2) --> Cl + NO3, with comparable yields. This experiment provides a direct measurement of the CIO product channel and consequently raises the possibility of an analogous channel in CIO dimer photolysis. Photodissociation of the CIO dimer is a critical step in the catalytic cycle that is presumed to dominate polar stratospheric ozone destruction. A substantial yield of CIO would reduce the efficiency of this cycle.

Photoprotection by carotenoids during photosynthesis: motional dependence of intramolecular energy transfer.

A new carotenoporphyrin has been prepared in which a synthetic carotenoid is joined to a tetraarylporphyrin through a flexible trimethylene linkage. This molecule exists primarily in an extended conformation with the carotenoid chromophore far from the porphyrin pi-electron system. In benzene solution, where large-amplitude molecular motions are rapid, the molecule can momentarily assume less stable conformations which favor triplet energy transfer, and quenching of the porphyrin triplet by the carotenoid is fast. In a polystyrene matrix or frozen glass such motions are slow, and energy transfer cannot compete with other pathways for depopulating the triplet state. These observations help establish the requirements for biological photoprotection.

Reproducible measurement of single-molecule conductivity.

A reliable method has been developed for making through-bond electrical contacts to molecules. Current-voltage curves are quantized as integer multiples of one fundamental curve, an observation used to identify single-molecule contacts. The resistance of a single octanedithiol molecule was 900 +/- 50 megohms, based on measurements on more than 1000 single molecules. In contrast, nonbonded contacts to octanethiol monolayers were at least four orders of magnitude more resistive, less reproducible, and had a different voltage dependence, demonstrating that the measurement of intrinsic molecular properties requires chemically bonded contacts.

Efficient multistep photoinitiated electron transfer in a molecular pentad.

A synthetic five-part molecular device has been prepared that uses a multistep electron transfer strategy similar to that of photosynthetic organisms to capture light energy and convert it to chemical potential in the form of long-lived charge separation. It consists of two covalently linked porphyrin moieties, one containing a zinc ion (P(Zn)) and the other present as the free base (P). The metailated porphyrin bears a carotenoid polyene (C) and the other a diquinone species (Q(A)-Q(B)). Excitation of the free-base porphyrin in a chloroform solution of the pentad yields an initial charge-separated state, C-P(Zn)-P(.+).-Q(A)(-)-Q(B), with a quantum yield of 0.85. Subsequent electron transfer steps lead to a final charge-separated state, C(.+)-P(Zn)-P-Q(A)-Q(B)(.-), which is formed with an overall quantum yield of 0.83 and has a lifetime of 55 microseconds. Irradiation of the free-base form of the pentad, C-P-P-Q(A)-Q(B), gives a similar charge-separated state with a lower quantum yield (0.15 in dichloromethane), although the lifetime is increased to approximately 340 microseconds. The artificial photosynthetic system preserves a significant fraction ( approximately 1.0 electron volt) of the initial excitation energy (1.9 electron volts) in the long-lived, charge-separated state.

Domain wall energy and strain in Pt/Co/Ir thin films on piezoelectric transducers.

We study the energy and creep velocity of magnetic domain walls in perpendicularly magnetised Pt/Co/Ir thin films under strain. We find that the enhancement of domain wall creep velocity under strain from piezoelectric transducers is largest in films with the thinnest Co layers (0.56 nm), in which the strain causes the smallest relative change in perpendicular magnetic anisotropy and the largest relative change in domain wall creep velocity. We show how domain wall energy is predictive of the sensitivity of domain wall creep velocity to changes in strain, and thus provide a route to designing magnetic thin film systems for optimum strain control.

Cytokine production and requirements during T-cell development.

Cytokines have been presumed to play an important role during T-cell development, but until recently formal proof has been missing. Most cytokine-knockout mice show apparently normal intrathymic T-cell development. The exception to this is IL-7, as both IL-7 and IL-7R alpha knockout mice show abnormally low thymic cellularity, making IL-7 the first cytokine shown to be necessary for normal intrathymic T-cell development.

Assessment of resuscitation as measured by markers of metabolic acidosis and features of injury.

AIMS: The best time for definitive orthopaedic care is often unclear in patients with multiple injuries. The objective of this study was make a prospective assessment of the safety of our early appropriate care (EAC) strategy and to evaluate the potential benefit of additional laboratory data to determine readiness for surgery. PATIENTS AND METHODS: A cohort of 335 patients with fractures of the pelvis, acetabulum, femur, or spine were included. Patients underwent definitive fixation within 36 hours if one of the following three parameters were met: lactate < 4.0 mmol/L; pH ≥ 7.25; or base excess (BE) ≥ -5.5 mmol/L. If all three parameters were met, resuscitation was designated full protocol resuscitation (FPR). If less than all three parameters were met, it was designated an incomplete protocol resuscitation (IPR). Complications were assessed by an independent adjudication committee and included infection; sepsis; PE/DVT; organ failure; pneumonia, and acute respiratory distress syndrome (ARDS). RESULTS: In total, 66 patients (19.7%) developed 90 complications. An historical cohort of 1441 patients had a complication rate of 22.1%. The complication rate for patients with only one EAC parameter at the point of protocol was 34.3%, which was higher than other groups (p = 0.041). Patients who had IPR did not have significantly more complications (31.8%) than those who had FPR (22.6%; p = 0.078). Regression analysis showed male gender and injury severity score to be independent predictors of complications. CONCLUSIONS: This study highlights important trends in the IPR and FPR groups, suggesting that differences in resuscitation parameters may guide care in certain patients; further study is, however, required. We advocate the use of the existing protocol, while research is continued for high-risk subgroups. Cite this article: Bone Joint J 2017;99-B:122-7.

IL-36γ is a crucial proximal component of protective type-1-mediated lung mucosal immunity in Gram-positive and -negative bacterial pneumonia.

Interleukin-36γ (IL-36γ) is a member of novel IL-1-like proinflammatory cytokine family that are highly expressed in epithelial tissues and several myeloid-derived cell types. Little is known about the role of the IL-36 family in mucosal immunity, including lung anti-bacterial responses. We used murine models of IL-36γ deficiency to assess the contribution of IL-36γ in the lung during experimental pneumonia. Induction of IL-36γ was observed in the lung in response to Streptococcus pneumoniae (Sp) infection, and mature IL-36γ protein was secreted primarily in microparticles. IL-36γ-deficient mice challenged with Sp demonstrated increased mortality, decreased lung bacterial clearance and increased bacterial dissemination, in association with reduced local expression of type-1 cytokines, and impaired lung macrophage M1 polarization. IL-36γ directly stimulated type-1 cytokine induction from dendritic cells in vitro in a MyD88-dependent manner. Similar protective effects of IL-36γ were observed in a Gram-negative pneumonia model (Klebsiella pneumoniae). Intrapulmonary delivery of IL-36γ-containing microparticles reconstituted immunity in IL-36γ-/- mice. Enhanced expression of IL-36γ was also observed in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome because of pneumonia. These studies indicate that IL-36γ assumes a vital proximal role in the lung innate mucosal immunity during bacterial pneumonia by driving protective type-1 responses and classical macrophage activation.

Pinning and hysteresis in the field dependent diameter evolution of skyrmions in Pt/Co/Ir superlattice stacks.

We have imaged Néel skyrmion bubbles in perpendicularly magnetised polycrystalline multilayers patterned into 1 µm diameter dots, using scanning transmission x-ray microscopy. The skyrmion bubbles can be nucleated by the application of an external magnetic field and are stable at zero field with a diameter of 260 nm. Applying an out of plane field that opposes the magnetisation of the skyrmion bubble core moment applies pressure to the bubble and gradually compresses it to a diameter of approximately 100 nm. On removing the field the skyrmion bubble returns to its original diameter via a hysteretic pathway where most of the expansion occurs in a single abrupt step. This contradicts analytical models of homogeneous materials in which the skyrmion compression and expansion are reversible. Micromagnetic simulations incorporating disorder can explain this behaviour using an effective thickness modulation between 10 nm grains.

Maternal variables associated with physiologic stress and perinatal complications in preterm infants.

OBJECTIVE: Complications of prematurity may be related to dysregulation of the hypothalamic-pituitary-adrenal axis in preterm infants. Increased intrauterine exposure to cortisol may be responsible for adverse prenatal programming and subsequent dysfunction of the infant's hypothalamic-pituitary-adrenal axis. The aim of the study was to describe maternal social variables and their association with infant cortisol levels and complications of prematurity. METHODS: Preterm infants <32 weeks' gestation were recruited. Primary outcomes were development of complications of prematurity and physiologic stress response, represented by cord blood and salivary cortisol levels on first day of life. Descriptive statistics and comparative analyses were performed. RESULTS: Fifteen of 31 infants enrolled developed a complication of prematurity. Infants of greater gestational age when prenatal care was established had lower cord blood cortisol (p = 0.009) and trended a higher risk of necrotizing enterocolitis (p = 0.069). Infants whose mothers smoked more showed significantly different salivary cortisol distributions on day 1 (p = 0.037), and were at greater risk for intraventricular hemorrhage (p = 0.018). CONCLUSIONS: The association between maternal social variables, hypothalamic-pituitary-adrenal axis dysregulation, and complications of prematurity supports the research model of physiologic dysregulation/allostatic load as a mechanism for complications in preterm infants. More research is warranted to investigate associations between maternal social variables, maternal stress levels, and adverse prenatal programming of the infant hypothalamic-pituitary-adrenal axis.

Is Early Appropriate Care of axial and femoral fractures appropriate in multiply-injured elderly trauma patients?

BACKGROUND: Previous work established resuscitation parameters that minimize complications with early fracture management. This Early Appropriate Care (EAC) protocol was applied to patients with advanced age to determine if they require unique parameters to mitigate complications. METHODS: Between October 2010 and March 2013, 376 consecutive skeletally mature patients with unstable fractures of the pelvis, acetabulum, thoracolumbar spine, and/or proximal or diaphyseal femur fractures were treated at a level I trauma center and were prospectively studied. Patients aged ≤30 years (n = 114), 30 to 60 years (n = 184), and ≥60 years (n = 37) with Injury Severity Scores (ISS) ≥16 and unstable fractures of the pelvis, acetabulum, spine, and/or diaphyseal femur were treated within 36 h, provided they showed evidence of adequate resuscitation. ISS, Glasgow Coma Scale (GCS), and American Society of Anesthesiologists (ASA) classification were determined. Lactate, pH, and base excess (BE) were measured at 8-h intervals. Complications included pneumonia, pulmonary embolism (PE), acute renal failure, acute respiratory distress syndrome (ARDS), multiple organ failure (MOF), deep vein thrombosis, infection, sepsis, and death. RESULTS: Patients ≤30 years old (y/o) were more likely to sustain gunshot wounds (p = 0.039), while those ≥60 y/o were more likely to fall from a height (p = 0.002). Complications occurred at similar rates for patients ≤30 y/o, 30 to 60 y/o, and ≥60 y/o. There were no differences in lactate, pH, or BE at the time of surgery. For patients ≤30 y/o, there were increased overall complications if pH was <7.30 (p = 0.042) or BE <-6.0 (p = 0.049); patients ≥60 y/o demonstrated more sepsis if BE was <-6.0 (p = 0.046). CONCLUSIONS: EAC aims to definitively manage axial and femoral shaft fractures once patients have been adequately resuscitated to minimize complications. EAC is associated with comparable complication rates in young and elderly patients. Further study is warranted with a larger sample to further validate EAC in elderly patients. LEVEL OF EVIDENCE: level II prospective, comparative study.

Modification of perpendicular magnetic anisotropy and domain wall velocity in Pt/Co/Pt by voltage-induced strain.

The perpendicular magnetic anisotropy K(eff), magnetization reversal, and field-driven domain wall velocity in the creep regime are modified in Pt/Co(0.85-1.0 nm)/Pt thin films by strain applied via piezoelectric transducers. K(eff), measured by the extraordinary Hall effect, is reduced by 10 kJ/m(3) by tensile strain out-of-plane ε(z) = 9 × 10(-4), independently of the film thickness, indicating a dominant volume contribution to the magnetostriction. The same strain reduces the coercive field by 2-4 Oe, and increases the domain wall velocity measured by wide-field Kerr microscopy by 30-100%, with larger changes observed for thicker Co layers. We consider how strain-induced changes in the perpendicular magnetic anisotropy can modify the coercive field and domain wall velocity.

Magnetic microscopy and topological stability of homochiral Néel domain walls in a Pt/Co/AlOx trilayer.

The microscopic magnetization variation in magnetic domain walls in thin films is a crucial property when considering the torques driving their dynamic behaviour. For films possessing out-of-plane anisotropy normally the presence of Néel walls is not favoured due to magnetostatic considerations. However, they have the right structure to respond to the torques exerted by the spin Hall effect. Their existence is an indicator of the interfacial Dzyaloshinskii-Moriya interaction (DMI). Here we present direct imaging of Néel domain walls with a fixed chirality in device-ready Pt/Co/AlOx films using Lorentz transmission electron and Kerr microscopies. It is shown that any independently nucleated pair of walls in our films form winding pairs when they meet that are difficult to annihilate with field, confirming that they all possess the same topological winding number. The latter is enforced by the DMI. The field required to annihilate these winding wall pairs is used to give a measure of the DMI strength. Such domain walls, which are robust against collisions with each other, are good candidates for dense data storage.

Intrapulmonary tumor necrosis factor gene therapy increases bacterial clearance and survival in murine gram-negative pneumonia.

Tumor necrosis factor alpha (TNF) has been shown to be an essential cytokine mediator of innate immunity in bacterial pneumonia. To augment the expression of TNF within the lung, a recombinant adenoviral vector containing the murine TNF cDNA (Ad5mTNF) has been developed, and the intratracheal administration of this vector resulted in the dose- and time-dependent expression of TNF in the lung, but not systemically. Administration of Ad5mTNF resulted in significant airspace and peribronchial inflammation, with a predominant neutrophil influx by 2 days, and mononuclear cell infiltrates by 4 to 7 days posttreatment. Importantly, the administration of Ad5mTNF at a dose of 1 x 10(8) PFU significantly improved the survival of animals challenged concomitantly with Klebsiella pneumoniae, which occurred in association with enhanced clearance of bacteria from the lung and decreased dissemination of K. pneumoniae to the bloodstream. However, the delivery of higher doses of Ad5mTNF (5 x 10(8) PFU) was not beneficial and in fact the intratracheal administration of a similar dose of control vector (Ad5LacZ) actually enhanced Klebsiella-induced lethality by impairing clearance of K. pneumoniae from the lung. Our studies suggests that the transient transgenic expression of TNF within the lung dose dependently augments antibacterial host defense in murine Klebsiella pneumonia.

Murine natural killer cell differentiation: past, present, and future.

Natural killer cells are bone marrow-derived lymphocytes capable of lysing a variety of target cells without prior exposure. While the biological activities and function of mature NK cells have been extensively investigated, the differentiation of NK cells from primitive hematopoietic stem cells is poorly understood. Recently, we have reported on the identification of a highly enriched bone marrow population capable of repopulating recipient mice with mature NK cells. In this review, we will summarize our findings and those of others in an attempt to clarify the current status of murine natural killer cell differentiation.