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The complete active space (CASSCF) and the multiconfiguration second-order perturbation theory (CASPT2) calculations with 6-311++G(3df,3pd) gauss basis sets are performed for several electronic states of the methylthio neutral radical and its cation and anion. Twenty-two electronic states are optimized in the

Significance Neurodegenerative diseases are poorly understood and difficult to treat. One common hallmark is lysosomal dysfunction leading to the accumulation of aggregates and other undegradable materials, which cause damage to brain resident cells. Lysosomes are acidic organelles responsible for breaking down biomolecules and recycling their constitutive parts. In this work, we find that the antiinflammatory and neuroprotective compound, discovered via a phenotypic screen, imparts its beneficial effects by targeting the lysosome and restoring its function. This is established using a genome-wide CRISPRi target identification screen and then confirmed using a variety of lysosome-targeted studies. The resulting small molecule from this study represents a potential treatment for neurodegenerative diseases as well as a research tool for the study of lysosomes in disease.

Description, An oligomeric nanozyme with ultrafast electron transfer can achieve high catalytic activity and alleviate acute brain injury., Artificial enzymes have attracted wide interest in disease diagnosis and biotechnology due to high stability, easy synthesis, and cost effectiveness. Unfortunately, their catalytic rate is limited to surface electron transfer, affecting the catalytic and biological activity. Here, we report an oligomeric nanozyme (O-NZ) with ultrafast electron transfer, achieving ultrahigh catalytic activity. O-NZ shows electron transfer of 1.8 nanoseconds in internal cores and 1.2 picoseconds between core and ligand molecule, leading to ultrahigh superoxidase dismutase–like and glutathione peroxidase–like activity (comparable with natural enzyme, Michaelis constant = 0.87 millimolars). Excitingly, O-NZ can improve the 1-month survival rate of mice with acute brain trauma from 50 to 90% and promote the recovery of long-term neurocognition. Biochemical experiments show that O-NZ can decrease harmful peroxide and superoxide via in vivo catalytic chain reaction and reduce acute neuroinflammation via nuclear factor erythroid-2 related factor 2–mediated up-regulation of heme oxygenase-1 expression.

Non-invasive deep-tissue three-dimensional optical imaging of live mammals with high spatiotemporal resolution is challenging owing to light scattering. We developed near-infrared II (1,000–1,700 nm) light-sheet microscopy with excitation and emission of up to approximately 1,320 nm and 1,700 nm, respectively, for optical sectioning at a penetration depth of approximately 750 μm through live tissues without invasive surgery and at a depth of approximately 2 mm in glycerol-cleared brain tissues. Near-infrared II light-sheet microscopy in normal and oblique configurations enabled in vivo imaging of live mice through intact tissue, revealing abnormal blood flow and T-cell motion in tumor microcirculation and mapping out programmed-death ligand 1 and programmed cell death protein 1 in tumors with cellular resolution. Three-dimensional imaging through the intact mouse head resolved vascular channels between the skull and brain cortex, and allowed monitoring of recruitment of macrophages and microglia to the traumatic brain injury site. Light-sheet microscopy in the NIR-II window enables rapid volumetric imaging of tissues at impressive depths in vivo without invasive preparations owing to the reduced light scattering and tissue autofluorescence at these wavelengths.

Microglia maintain homeostasis in the central nervous system through phagocytic clearance of protein aggregates and cellular debris. This function deteriorates during ageing and neurodegenerative disease, concomitant with cognitive decline. However, the mechanisms of impaired microglial homeostatic function and the cognitive effects of restoring this function remain unknown. We combined CRISPR-Cas9 knockout screens with RNA sequencing analysis to discover age-related genetic modifiers of microglial phagocytosis. These screens identified CD22, a canonical B cell receptor, as a negative regulator of phagocytosis that is upregulated on aged microglia. CD22 mediates the anti-phagocytic effect of α2,6-linked sialic acid, and inhibition of CD22 promotes the clearance of myelin debris, amyloid-β oligomers and α-synuclein fibrils in vivo. Long-term central nervous system delivery of an antibody that blocks CD22 function reprograms microglia towards a homeostatic transcriptional state and improves cognitive function in aged mice. These findings elucidate a mechanism of age-related microglial impairment and a strategy to restore homeostasis in the ageing brain.

The difluoroamino derivatives of prismane were designed and calculated at the B3LYP/6-311G** level. The detonation performances, as well as the band gap, were investigated to look for high energy density compounds. Our calculations showed that difluoroamino group is an effective substitute group for increasing density and heats of formation. All compounds have large heats of formation, and there is a linear relationship between the heats of formation (HOFs) and the substituent numbers. Bond dissociation energies of trigger bonds are all over 200kJ/mol, which indicated that these prismane derivatives have good stability. More than three substituted groups made the detonation performances meet the requirement of energetic materials. Our calculations provided basic information for the further syntheses of title molecules.

Traumatic brain injury (TBI) is a leading cause of long-term neurological disability. Currently there is no effective pharmacological treatment for patients suffering from the long-lasting symptoms of TBI. We recently discovered that colony stimulating factor 1 (CSF1), an essential regulator of macrophage homeostasis, is neuroprotective and reduces neuroinflammation in two models of neurological disease in mice. Here we used a mouse model of repetitive mild TBI (mTBI) to examine whether CSF1 would attenuate cognitive deficits and improve pathological outcomes in two paradigms. In the acute paradigm, a single bolus treatment of CSF1 administered 24 h after injury significantly reduces memory impairment and astrocyte reactivity assessed 3 months later. In the chronic paradigm, the mice were tested 3 months after mTBI when they showed cognitive deficits. The mice were then randomly assigned to receive CSF1 or PBS (as control) treatment. After one month of treatment, the PBS-treated mice remained cognitively impaired, but the CSF1-treated showed significant improvements in cognitive function. RNA-seq and Ingenuity Pathway Analysis reveals CSF1 treatment alters cognition- and memory-related transcriptomic changes and pathways. The results of this study show that acute as well as delayed CSF1 treatment attenuate chronically impaired cognitive functions and improve pathological outcomes long after mTBI. The wide therapeutic time window of CSF1, together with the fact that CSF1 is approved for human use in clinical trials, strongly supports the potential clinical usefulness of this treatment in patients with mTBI.

By replacing hydrogen atoms in thymine molecules with nitro groups, a series of new high-energy-density molecules are designed. To explore the thermal stability, the heats of formation (HOF) are calculated at the B3PW91-D3/6-311++G(2df,2p) level. The bond dissociation energy and the bond order are also calculated to predict the kinetic stability at the same level. Based on our calculations, excellent stability is confirmed for title molecules. To confirm the possibility of application as high-energy-density compounds, the molecular density (ρ), explosive heats (Q), detonation velocity (D), detonation pressure (P), free space per molecule in crystal lattice (ΔV), and characteristic drop height (H50) are calculated. On the consideration of the stability and the detonation characters, E1 is confirmed as the candidates of high-energy-density compounds.

The vascular interface of the brain, known as the blood–brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability1–3. Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins4,5. Thus, it is unclear whether permeability to individually injected exogenous tracers—as is standard in BBB studies—fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery. Tagging and tracking the blood plasma proteome as a discovery tool reveals widespread endogenous transport of proteins into the healthy brain and the pharmacologically modifiable mechanisms by which the brain endothelium regulates this process with age.

Nitro-substituted derivatives of hypoxanthine are designed by substituting the hydrogen atoms by nitro groups. The heats of formation (HOF) are calculated at the G3MP2 level to confirm the thermal stability. Also, the bond dissociation energy (BDE) accompanied by the bond order is calculated at the same level to explore kinetic stability. To evaluate the potential application as high-energy density materials, the explosive heat, the molecular density, the detonation pressure, and detonation velocity are estimated by using the Kamlet-Jacobs (K-J) equation. Furthermore, the molecular sensitivities are considered by calculating the character height (H50) and the amount of free space per molecule in the crystal lattice (ΔV). Based on our calculations, the trinitro-substituted hypoxanthine (C) is screened out as the potential high-energy density compounds for further study.

A series of nitramino-derivatives of pyrrole were designed through introducing the nitramino group into pyrrole ring to look for high-energy-density compounds. The molecular stabilities are explored thoroughly based on the calculated heats of formation, bond dissociation energy, and bond order at the B3PW91/6-311+G(d,p) level. The results indicated that the molecules designed in this paper have enough stability not only thermally but also kinetically. To evaluate the potential application as high-energy-density compounds, the detonation velocity and detonation pressure are calculated by using the Kamlet-Jacobs equation. Based on the calculation both of stability and detonation characters, four nitramino-derivatives of pyrrole (D1: 2,3-trinitramino-1H-pyrrole, D2: 2,3,4-trinitramino-1H-pyrrole, D3: 2,3,5-trinitramino-1H- pyrrole and E: 2,3,4,5- tetranitramino- 1H-pyrrole) are screened out as potential high-energy-density molecules for further study.

A series of derivatives of nitroso-substituted cubane were designed through the substitution of hydrogen atoms by nitroso groups one by one. The heats of formation (HOFs) were calculated to explore the thermal stability. The bond dissociation energies (BDEs) and the bond orders of the trigger bonds were also investigated to explore the molecular stability kinetically. Furthermore, the steric effect was confirmed as the determinant of molecular stability for title molecules. To explore the detonation properties, the detonation pressure (P), the detonation velocity (D), the heat of detonation (Q), and the specific density (ρ) were calculated by using the empirical Kamlet-Jacobs (K-J) equations. To predict the sensitivity, the characteristic drop height (H50) is calculated. Based on our calculations, the octanitrosocubane is found with detonation parameters (D = 9.03 km/s, P = 36.96 GPa) better than that of RDX with enough stability.

Fluorescence imaging of biological systems in the second near-infrared (NIR-II, 1000–1700 nm) window has shown promise of high spatial resolution, low background, and deep tissue penetration owing to low autofluorescence and suppressed scattering of long wavelength photons. Here we develop a bright organic nanofluorophore (named p-FE) for high-performance biological imaging in the NIR-II window. The bright NIR-II >1100 nm fluorescence emission from p-FE affords non-invasive in vivo tracking of blood flow in mouse brain vessels. Excitingly, p-FE enables one-photon based, three-dimensional (3D) confocal imaging of vasculatures in fixed mouse brain tissue with a layer-by-layer imaging depth up to ~1.3 mm and sub-10 µm high spatial resolution. We also perform in vivo two-color fluorescence imaging in the NIR-II window by utilizing p-FE as a vasculature imaging agent emitting between 1100 and 1300 nm and single-walled carbon nanotubes (CNTs) emitting above 1500 nm to highlight tumors in mice., Imaging in the second near-infrared window has attracted attention due to superior penetration depth and low signal interference. Here, the authors describe a new organic nano fluorophore with high quantum yield and demonstrate its use for in vivo imaging.

Czirr et al. report that microglia lacking complement receptor 3 display increased extracellular Aβ degrading activity and that targeting the receptor with a small molecule increases Aβ clearance in vivo, thus identifying a microglial receptor as a novel therapeutic target., Recent genetic evidence supports a link between microglia and the complement system in Alzheimer’s disease (AD). In this study, we uncovered a novel role for the microglial complement receptor 3 (CR3) in the regulation of soluble β-amyloid (Aβ) clearance independent of phagocytosis. Unexpectedly, ablation of CR3 in human amyloid precursor protein–transgenic mice results in decreased, rather than increased, Aβ accumulation. In line with these findings, cultured microglia lacking CR3 are more efficient than wild-type cells at degrading extracellular Aβ by secreting enzymatic factors, including tissue plasminogen activator. Furthermore, a small molecule modulator of CR3 reduces soluble Aβ levels and Aβ half-life in brain interstitial fluid (ISF), as measured by in vivo microdialysis. These results suggest that CR3 limits Aβ clearance from the ISF, illustrating a novel role for CR3 and microglia in brain Aβ metabolism and defining a potential new therapeutic target in AD.

A series of nitro-imidazole derivatives were designed by replacing hydrogen atoms on imidazole ring with nitro group one by one. In order to investigate the thermodynamic stability, heat of formation (HOF), and bond dissociation energy (BDE) are calculated at the B3PW91/6-311+G(d,p) level. In order to investigate the impact sensitivity and detonation property, the drop height (H50), free space per molecule in crystal lattice (ΔV), detonation velocity (D), and detonation pressure (P) are calculated by using the empirical Kamlet–Jacobs (K-J) equation. The results show that the thermal stabilities of title molecules are determined by whether nitro group is associated to 1-position or not and accompanied with the steric hindrance between nitro groups and the charge population on the carbon atoms of imidazole ring. The excellent impact sensitivity and detonation performance of title molecules are also evaluated. On the consideration both of stability and detonation characters, 2,4,5-trinitro-1H-imidazole (D = 8.98 km/s, P = 36.70 GPa) is screened out as the potential high-energy-density molecule for further research.

A series of derivatives of pyridine were designed through substituting hydrogen atoms by nitro groups systematically. By using the density functional theory at B3PW91/6-311++G(d,p)//MP2/311++G(d,p) level, heats of formation, bond orders, and bond dissociation energies were calculated to explore the thermodynamic stabilities of title molecules. Furthermore, the regularity of stability was explained based on the electronic population. Our results indicated that title molecules had enough stability to exist. To evaluate the potential usage as a high-energy-density molecule, the detonation pressure and detonation velocity were explored by using the semi-empirical Kamlet-Jacobs equation and excellent detonation character was confirmed. Overall consideration of the thermal stability and energetic character, four molecules (2,3,4,5-tetranitropyridine, 2,3,5,6-tetranitropyridine, 2,4,5,6-tetranitrop-pyridine, 2,3,4,5,6-pentanitropyridine) were confirmed to be better than RDX and filtered as potential energetic molecules.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide, therefore important to understanding pathogenesis. Here, we designed a bright, renal-excreted and biocompatible near infrared II molecular fluorophore for in vivo imaging of TBI. We observed a transient hypoperfusion in the injured cerebral region, followed by fluorophore leakage. NIR-II fluorophores can provide a non-invasive assessment to TBI.

Spectrally selective materials are of great interest for optoelectronic devices in which wavelength-selectivity of the photoactive material is necessary for applications such as multi-junction solar cells, narrow-band photodetectors, transparent photovoltaics, and tailored emission sources. Achieving controlled transparency or opacity within multiple wavelength bands in the absorption, reflection, and transmission spectra are difficult to achieve in traditional semiconductors that typically absorb at all energies above their electronic band gap and is generally realized by the use of external bandpass filters. Here, we propose an alternate method for achieving spectral selectivity in optoelectronic thin films: the use of photonic band engineering within the absorbing region of a semiconductor in which resonant photonic bands are strongly coupled to the external reflectivity and transmission spectra. As a first step, we use optical simulations to systematically study the effect of material absorption on the properties of the photonic bands in a photonic crystal slab structure. We find that adding a weak loss to the materials model does not appreciably change the frequencies of the photonic bands but does reduce the quality factor of the associated photonic modes. Critically, the radiating photonic bands induce strong Fano resonance features in the transmission and reflection spectra, even in the presence of material absorption, due to coupling between the bands and external electromagnetic plane waves. These resonances can be tuned by adjusting the photonic crystal structural properties to induce spectral selectivity in the absorbing region of semiconductors. Lastly, we demonstrate this tuning method experimentally by fabricating a proof-of-principle photonic structure consisting of a self-assembled polystyrene bead monolayer infiltrated with PbS CQDs that displays both near-infrared absorption enhancement and visible transparency enhancement over a homogeneous control film, qualitatively matching predictions and showing promise for optoelectronic applications.

Microglia maintain homeostasis in the central nervous system through phagocytic clearance of protein aggregates and cellular debris. This function deteriorates during ageing and neurodegenerative disease, concomitant with cognitive decline. However, the mechanisms of impaired microglial homeostatic function and the cognitive effects of restoring this function remain unknown. We combined CRISPR-Cas9 knockout screens with RNA sequencing analysis to discover age-related genetic modifiers of microglial phagocytosis. These screens identified CD22, a canonical B cell receptor, as a negative regulator of phagocytosis that is upregulated on aged microglia. CD22 mediates the anti-phagocytic effect of α2,6-linked sialic acid, and inhibition of CD22 promotes the clearance of myelin debris, amyloid-β oligomers and α-synuclein fibrils in vivo. Long-term central nervous system delivery of an antibody that blocks CD22 function reprograms microglia towards a homeostatic transcriptional state and improves cognitive function in aged mice. These findings elucidate a mechanism of age-related microglial impairment and a strategy to restore homeostasis in the ageing brain.

An amphiphilic brush block copolymer P(St-g-PBDEMA)-b-P(MEO2MA-co-OEGMA) was prepared by ATRP, RAFT and click chemistry. The copolymer micelles show temperature and glucose responses. The controlled release of insulin could be achieved by adjusting temperature and glucose concentration.

Transforming growth factor-β (TGF-β) plays an important role in the development and maintenance of embryonic dopaminergic (DA) neurons in the midbrain. To study the function of TGF-β signaling in the adult nigrostriatal system, we generated transgenic mice with reduced TGF-β signaling in mature neurons. These mice display age-related motor deficits and degeneration of the nigrostriatal system. Increasing TGF-β signaling in the substantia nigra through adeno-associated virus expressing a constitutively active type I receptor significantly reduces 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration and motor deficits. These results suggest that TGF-β signaling is critical for adult DA neuron survival and that modulating this signaling pathway has therapeutic potential in Parkinson disease.SIGNIFICANCE STATEMENTWe show that reducing Transforming growth factor-β (TGF-β) signaling promotes Parkinson disease-related pathologies and motor deficits, and increasing TGF-β signaling reduces neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a parkinsonism-inducing agent. Our results provide a rationale to pursue a means of increasing TGF-β signaling as a potential therapy for Parkinson's disease.

To determine whether steroid avoidance in pediatric kidney transplantation is safe and efficacious, a randomized, multicenter trial was performed in 12 pediatric kidney transplant centers. One hundred thirty children receiving primary kidney transplants were randomized to steroid-free (SF) or steroid-based (SB) immunosuppression, with concomitant tacrolimus, mycophenolate and standard dose daclizumab (SB group) or extended dose daclizumab (SF group). Follow-up was 3 years posttransplant. Standardized height Z-score change after 3 years follow-up was -0.99 ± 2.20 in SF versus -0.93 ± 1.11 in SB; p = 0.825. In subgroup analysis, recipients under 5 years of age showed improved linear growth with SF compared to SB treatment (change in standardized height Z-score at 3 years -0.43 ± 1.15 vs. -1.07 ± 1.14; p = 0.019). There were no differences in the rates of biopsy-proven acute rejection at 3 years after transplantation (16.7% in SF vs. 17.1% in SB; p = 0.94). Patient survival was 100% in both arms; graft survival was 95% in the SF and 90% in the SB arms (p = 0.30) at 3 years follow-up. Over the 3 year follow-up period, the SF group showed lower systolic BP (p = 0.017) and lower cholesterol levels (p = 0.034). In conclusion, complete steroid avoidance is safe and effective in unsensitized children receiving primary kidney transplants.

To identify biomarkers of operational tolerance in pediatric and adult liver transplant recipients, transcriptional profiles were examined from 300 samples by microarrays and Q-PCR measurements of blood specimens from pediatric and adult liver transplant recipients and normal tissues. Tolerance-specific genes were validated in independent samples across two different transplant programs and validated by Q-PCR. A minimal set of 13 unique genes, highly expressed in natural killer cells (p = 0.03), were significantly expressed in both pediatric and adult liver tolerance, irrespective of different clinical and demographic confounders. The performance of this gene set by microarray in independent samples was 100% sensitivity and 83% specificity and the AUC was 0.988 for only three genes by Q-PCR. 26% of adults and 64% of children with excellent liver allograft function, on minimal or dual immunosuppression, showed high prediction scores for tolerance. Novel peripheral transcriptional profiles can be identified in operational tolerance in pediatric and adult recipients of liver allografts, suggesting a high incidence of a pro-tolerogenic phenotype in stable patients on chronic immunosuppression. Given the high incidence of viral infections and malignancies in liver transplant recipients, this gene set provides an important monitoring tool that can move the field toward personalized and predictive medicine in organ transplantation.

The nucleotide sequence of the genome of the nucleopolyhedrovirus (NPV) from Mamestra configurata (MacoNPV, isolate 90/2), a group II NPV, was determined and analyzed. The MacoNPV DNA genome consists of 155,060 bp and has an overall G+C content of 41.7%. Computer-assisted analysis predicted 169 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. BLAST searches and comparisons with completely sequenced baculoviruses indicated that there were 66 ORFs conserved among the nine baculoviruses compared and an additional 17 ORFs were conserved among the NPVs. The gene content and gene arrangement in MacoNPV were most similar to those of SeMNPV, including two putative odv-e66 and p26 gene homologues. However, in contrast to SeMNPV, 8 ORFs with homology to baculovirus repeat ORFs (bro) and single copies of enhancin and conotoxin-like protein ORFs were found in MacoNPV. The MacoNPV genome contained four homologous regions, each with 10 to 17 repeated sequences. Each repeat was 60 to 86 nucleotides in length and contained an approximately 43-bp-long imperfect palindrome. There were 13 ORFs unique to MacoNPV, ranging from a small ORF of 196 bp to larger ORFs of up to 1047 bp, and many of these contained typical early and late baculovirus consensus promoters.

The late expression factor-5 gene ( lef-5 ) of Autographa californica multinucleocapsid polyhedrovirus (AcMNPV) is required for late gene expression. In this paper, we demonstrate that LEF-5 interacts with itself in the yeast two-hybrid system and in glutathione–S-transferase affinity assays. Deletion analysis suggested that the C-terminal 71 amino acids (aa) were not required for interaction. However, all deletions tested involving the N-terminal 194 aa significantly reduced LEF-5:LEF-5 interaction. LEF-5 or LEF-5 deletion mutants were transfected into Sf-9 cells with the full complement of genes required for baculovirus late transcription. All deletion clones tested reduced expression of a β-glucuronidase (GUS) reporter gene under control of the late vp39 capsid promoter. Amino-acid sequence analysis of LEF-5 identified a previously unreported domain within the C-terminal 32 aa that is homologous to the zinc ribbon domain of RNA polymerase II elongation factor IIS (TFIIS) from a variety of taxa. Molecular modeling of the putative LEF-5 Zn ribbon using the NMR data available for the Zn ribbon of TFIIS suggested that this domain could fold into a Zn ribbon structure similar to TFIIS. Alanine scanning mutagenesis of amino acids predicted to be important for functioning of the LEF-5 ribbon structure significantly reduced LEF-5 activity in transient expression assays. Mutations changing the amino acids predicted to coordinate Zn 2+ caused a reduction in activity similar to that when the domain was eliminated completely.

We report 1-year outcomes of a randomized study of Rituximab versus standard-of-care immunosuppression (Thymoglobulin and/or pulse steroids) for treatment of biopsy confirmed, acute transplant rejection with B-cell infiltrates, in 20 consecutive recipients (2-23 years). Graft biopsies, with Banff and CADI scores, CD20 and C4d stains, were performed at rejection and 1 and 6 months later. Peripheral blood CMV, EBV and BK viral loads, graft function, DSA, immunoglobulins, serum humanized antichimeric antibody (HACA) and Rituximab, and lymphocyte counts were monitored until 1 year posttreatment. Rituximab infusions were given with a high index of safety without HACA development and increased infections complications. Rituximab therapy resulted in complete tissue B-cell depletion and rapid peripheral B-cell depletion. Peripheral CD19 cells recovered at a mean time of approximately 12 months. There were some benefits for the recovery of graft function (p = 0.026) and improvement of biopsy rejection scores at both the 1- (p = 0.0003) and 6-month (p0.0001) follow-up biopsies. Reappearance of C4d deposition was not seen on follow-up biopsies after Rituximab therapy, but was seen in 30% of control patients. There was no change in DSA in either group, independent of rejection resolution. This study reports safety and suggests further investigation of Rituximab as an adjunctive treatment for B-cell-mediated graft rejection.

A second genotype of Mamestra configurata nucleopolyhedrovirus-A (MacoNPV-A), variant 90/4 (v90/4), was identified due to its altered restriction endonuclease profile and reduced virulence for the host insect, M. configurata, relative to the archetypal genotype, MacoNPV-A variant 90/2 (v90/2). To investigate the genetic differences between these two variants, the genome of v90/4 was sequenced completely. The MacoNPV-A v90/4 genome is 153 656 bp in size, 1404 bp smaller than the v90/2 genome. Sequence alignment showed that there was 99·5 % nucleotide sequence identity between the genomes of v90/4 and v90/2. However, the v90/4 genome has 521 point mutations and numerous deletions and insertions when compared to the genome of v90/2. Gene content and organization in the genome of v90/4 is identical to that in v90/2, except for an additional bro gene that is found in the v90/2 genome. The region between hr1 and orf31 shows the greatest divergence between the two genomes. This region contains three bro genes, which are among the most variable baculovirus genes. These results, together with other published data, suggest that bro genes may influence baculovirus genome diversity and may be involved in recombination between baculovirus genomes. Many ambiguous residues found in the v90/4 sequence also reveal the presence of 214 sequence polymorphisms. Sequence analysis of cloned HindIII fragments of the original MacoNPV field isolate that the 90/4 variant was derived from indicates that v90/4 is an authentic variant and may represent approximately 25 % of the genotypes in the field isolate. These results provide evidence of extensive sequence variation among the individual genomes comprising a natural baculovirus outbreak in a continuous host population.

Enhancin genes have been identified in a number of baculoviruses and enhancin proteins are characterized by their ability to enhance the oral infectivity of heterologous baculoviruses in various lepidopteran insects. Here, we describe the putative enhancin gene from Mamestra configurata nucleopolyhedrovirus (MacoNPV), only the second NPV in which an enhancin-like ORF has been identified. The putative enhancin gene from MacoNPV has a typical baculovirus late promoter (ATAAG) 15 bp upstream from the ATG codon. The enhancin ORF encodes an 847 amino acid protein with a predicted molecular mass of 98 kDa and contains a conserved zinc-binding domain (HEIAH) common to metalloproteases. The MacoNPV enhancin shows approximately 20 % amino acid identity with other baculovirus enhancins. An Autographa californica M nucleopolyhedrovirus (AcMNPV) recombinant, AcMNPV-enMP2, expressing the MacoNPV enhancin gene under control of its native promoter was developed and characterized. Northern blot analysis showed expression of enhancin from 24 through 72 h post-infection. In 2nd-instar Trichoplusia ni larvae, the LD50 of the AcMNPV-enMP2 recombinant was 2.8 polyhedral inclusion bodies (PIB) per larva, 4.4 times lower than that of AcMNPV E2 wild-type virus (12.4 PIB per larva). At biologically equivalent doses, i.e. LD90, the survival time 50 % (ST50) of AcMNPV-enMP2 recombinant and AcMNPV E2 wild-type viruses were not significantly different.

All baculovirus genomes sequenced to date encode a homolog of an alkaline nuclease that has been characterized in the Herpesviridae . In this report we describe the characterization of the alkaline nuclease (AN) homolog of the Autographa californica multinucleocapsid nucleopolyhedrovirus (Ac M NPV) (open reading frame 133). His-tagged AN constructs were expressed in recombinant baculoviruses and affinity purified, and then their enzymatic activity was characterized. AN was found to degrade linear DNA at alkaline pH, preferred Mg 2+ over Mn 2+ , had optimal activity at 35°C, and did not appear to have a salt requirement. To rule out contamination by the endogenous baculovirus gene product or a cellular enzyme, point mutations were introduced into a highly conserved domain of the gene. These mutations were found to markedly reduce or eliminate most of the activity of the affinity-purified enzyme. An antibody generated against the protein was used to analyze its expression by Western blot analysis. AN was found to be expressed at low levels by 12 h postinfection, with maximal expression at 24 h postinfection. Attempts to generate a virus with this gene inactivated were unsuccessful, suggesting that AN may be encoded by an essential gene.

We were unable to confirm transient late gene expression using constructs of 18 genes that had been reported to support Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) late gene expression when transfected into Spodoptera frugiperda cells [Lu, A., and Miller, L. K. (1995). J. Virol. 69, 975–982]. Three genes ( orf66, orf68, and orf41 ) were included, all or in part, in the constructs used in that study, but they had not been independently tested. Therefore we investigated these and neighboring orf s for their influence on late gene expression. We found that orf41 was required for late gene expression and that sequences within orf45 appeared to be required for the expression of orf41 . Although orf66 and orf68 did not appear to affect late gene expression, orf69 stimulated expression. orf69 was found to have high homology to recent entries in GenBank from a variety of organisms. In addition, it was found that orf121 , which was shown to be involved in early gene expression, and the viral homolog of pcna did not influence late gene expression.

Background Moyamoya Disease is a rare, devastating cerebrovascular disorder characterized by stenosis/occlusion of supraclinoid internal carotid arteries and development of fragile collateral vessels. Moyamoya Disease is typically diagnosed by angiography after clinical presentation of cerebral hemorrhage or ischemia. Despite unclear etiology, previous reports suggest there may be an immunological component. Methods To explore the role of autoimmunity in moyamoya disease, we used high-density protein arrays to profile IgG autoantibodies from the sera of angiographically-diagnosed Moyamoya Disease patients and compared these to healthy controls. Protein array data analysis followed by bioinformatics analysis yielded a number of auto-antibodies which were further validated by ELISA for an independent group of MMD patients (n = 59) and control patients with other cerebrovascular diseases including carotid occlusion, carotid stenosis and arteriovenous malformation. Results We identified 165 significantly (p Conclusions We report the first high-throughput analysis of autoantibodies in Moyamoya Disease, the results of which may provide valuable insight into the immune-related pathology of Moyamoya Disease and may potentially advance diagnostic clinical tools.

MacoNPV-96B is a nucleopolyhedrovirus isolated from naturally infected Mamestra configurata (Lepidoptera: Noctuidae) larvae. It was initially identified due to its completely different restriction endonuclease profile relative to the previously sequenced Mamestra configurata virus MacoNPV-90/2 (Q. Li, C. Donly, L. Li, L. G. Willis, D. A. Theilmann, and M. Erlandson, 2002, Virology 294, 106–121). The MacoNPV-96B host range and virulence were also found to differ significantly from those of the previous isolate. To further understand the complex of viruses infecting M. configurata, the genome of MacoNPV-96B was completely sequenced and analyzed in comparison with the genome of MacoNPV-90/2 and other sequenced baculoviruses. MacoNPV-96B consists of 158,482 bp, and 168 open reading frames (ORFs) of 150 nucleotides or longer with minimal overlap have been identified. The genome of MacoNPV-96B is 3422 bp larger than MacoNPV-90/2 and although gene arrangement is virtually identical, there are 9 ORFs unique to MacoNPV-96B and 10 unique to MacoNPV-90/2. bro genes were found to be associated with nonhomologous regions, suggesting that bro genes may facilitate recombination between genomes. A major difference in the gene content between the two viruses is a 5.4-kb insert in MacoNPV-96B, which is highly homologous to a cluster of Xestia c-nigrum granulovirus (XecnGV) ORFs, suggesting recent recombination events between these two viruses. Nucleotide sequence and amino acid sequence identity between the common ORFs of MacoNPV-96B and MacoNPV-90/2 average 87 and 90%, respectively. The sequence data suggest that MacoNPV-96B and MacoNPV-90/2 are closely related but have diverged and evolved into two separate species. This is the first study to identify highly related but separately evolving viruses in the same insect host and geographic location. A new Identity-GeneParityPlot analysis was developed to perform a comparison of two viral genomes in gene content and arrangement as well as homology level of individual ORFs.

IE0 is the only known baculovirus protein that is produced by splicing. In this study, we have explored the role of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) IE0 and its interaction with IE1 in the activation of very late gene expression from the polyhedrin promoter using transient assays. IE0 is co-expressed with IE1 throughout infection up to late times post-infection (p.i.) but shows peak levels of expression at early times. Significant changes in the ratios of the relative levels of IE0 to IE1 were observed throughout the course of infection. To study IE0 in the absence of IE1, we constructed a plasmid pAc-IE0(M-->A) that expressed only IE0. This was due to a mutation of the internal AUG that prevented translation of IE1 from the ie0 mRNA. Both IE0 and IE0(M-->A) were able to replace IE1 in transient assays, showing that IE0 is functional for very late gene activation and should be considered the 20th late gene expression factor (lef). In transient assays, IE0 showed that maximum very late gene expression is achieved at very low relative levels of protein. In contrast, IE1 requires higher levels of protein to obtain maximum very late gene expression. Furthermore, when the levels of IE0 become too high, very late gene expression rapidly declines. Interestingly, co-expression of IE0 and IE1 results in a mutually antagonistic affect on very late gene expression.

The degree of progressive chronic histological damage is associated with long-term renal allograft survival. In order to identify promising molecular targets for timely intervention, we examined renal allograft protocol and indication biopsies from 120 low-risk pediatric and adolescent recipients by whole-genome microarray expression profiling. In data-driven analysis, we found a highly regulated pattern of adaptive and innate immune gene expression that correlated with established or ongoing histological chronic injury, and also with development of future chronic histological damage, even in histologically pristine kidneys. Hence, histologically unrecognized immunological injury at a molecular level sets the stage for the development of chronic tissue injury, while the same molecular response is accentuated during established and worsening chronic allograft damage. Irrespective of the hypothesized immune or nonimmune trigger for chronic allograft injury, a highly orchestrated regulation of innate and adaptive immune responses was found in the graft at the molecular level. This occurred months before histologic lesions appear, and quantitatively below the diagnostic threshold of classic T-cell or antibody-mediated rejection. Thus, measurement of specific immune gene expression in protocol biopsies may be warranted to predict the development of subsequent chronic injury in histologically quiescent grafts and as a means to titrate immunosuppressive therapy.