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Background: Mild traumatic brain injury (mTBI) generally resolves within weeks. However, 15-30% of patients present persistent pathological and neurobehavioral sequelae that negatively affect their quality of life. Hyperhomocysteinemia (HHCY) is a neurotoxic condition derived from homocysteine accumulation above 15 μM. HHCY can occur in diverse stressful situations, including those sustained by U.S. active-duty service members on the battlefield or during routine combat practice. Mild-TBI accounts for more than 80% of all TBI cases, and HHCY exists in 5-7% of the general population. We recently reported that moderate HHCY exacerbates mTBI-induced cortical injury pathophysiology, including increased oxidative stress. Several studies have demonstrated hippocampus vulnerability to oxidative stress and its downstream effects on inflammation and cell death., Objective: This study aimed to assess the deleterious impact of HHCY on mTBI-associated hippocampal pathological changes. We tested the hypothesis that moderate HHCY aggravates mTBI-induced hippocampal pathological changes., Methods: HHCY was induced in adult male Sprague-Dawley rats with a high methionine dose. Rats were then subjected to mTBI by controlled cortical impact under sustained HHCY. Blood plasma was assessed for homocysteine levels and brain tissue for markers of oxidative stress, blood-brain barrier integrity, and cell death. Endothelial cell ultrastructure was assessed by Electron Microscopy and working memory performance using the Y maze test., Results: HHCY increased the hippocampal expression of nitrotyrosine in astroglial cells and decreased tight junction protein occludin levels associated with the enlargement of the endothelial cell nucleus. Furthermore, HHCY altered the expression of apoptosis-regulating proteins α-ii spectrin hydrolysis, ERK1/2, and AKT phosphorylation, mirrored by exacerbated mTBI-related hippocampal neuronal loss and working memory deficits., Conclusion: Our findings indicate that HHCY is an epigenetic factor that modulates mTBI pathological progression in the hippocampus and represents a putative therapeutic target for mitigating such physiological stressors that increase severity., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2023.)

The proinflammatory state associated with diabetes mellitus (DM) remains poorly understood. We found patients with DM have 3- to 14-fold elevations of blood-borne microparticles (MPs) that bind phalloidin (Ph; Ph positive [+] MPs), indicating the presence of F-actin on their surface. We hypothesized that F-actin-coated MPs were an unrecognized cause for DM-associated proinflammatory status. Ph+MPs, but not Ph-negative MPs, activate human and murine (Mus musculus) neutrophils through biophysical attributes of F-actin and membrane expression of phosphatidylserine (PS). Neutrophils respond to Ph+MPs via a linked membrane array, including the receptor for advanced glycation end products and CD36, PS-binding membrane receptors. These proteins in conjunction with TLR4 are coupled to NO synthase 1 adaptor protein (NOS1AP). Neutrophil activation occurs because of Ph+MPs causing elevations of NF-κB and Src kinase (SrcK) via a concurrent increased association of NO synthase 2 and SrcK with NOS1AP, resulting in SrcK S-nitrosylation. We conclude that NOS1AP links PS-binding receptors with intracellular regulatory proteins. Ph+MPs are alarmins present in normal human plasma and are increased in those with DM and especially those with DM and a lower-extremity ulcer., (Copyright © 2023 The Authors.)

Autophagy is a cellular catabolic pathway generally thought to be neuroprotective. However, autophagy and in particular its upstream regulator, the ULK1 kinase, can also promote axonal degeneration. We examined the role and the mechanisms of autophagy in axonal degeneration using a mouse model of contusive spinal cord injury (SCI). Consistent with activation of autophagy during axonal degeneration following SCI, autophagosome marker LC3, ULK1 kinase, and ULK1 target, phospho-ATG13, accumulated in the axonal bulbs and injured axons. SARM1, a TIR NADase with a pivotal role in axonal degeneration, colocalized with ULK1 within 1 h after SCI, suggesting possible interaction between autophagy and SARM1-mediated axonal degeneration. In our in vitro experiments, inhibition of autophagy, including Ulk1 knockdown and ULK1 inhibitor, attenuated neurite fragmentation and reduced accumulation of SARM1 puncta in neurites of primary cortical neurons subjected to glutamate excitotoxicity. Immunoprecipitation data demonstrated that ULK1 physically interacted with SARM1 in vitro and in vivo and that SAM domains of SARM1 were necessary for ULK1-SARM1 complex formation. Consistent with a role in regulation of axonal degeneration, in primary cortical neurons ULK1-SARM1 interaction increased upon neurite damage. Supporting a role for autophagy and ULK1 in regulation of SARM1 in axonal degeneration in vivo, axonal ULK1 activation and accumulation of SARM1 were both decreased after SCI in Becn1 +/- autophagy hypomorph mice compared to wild-type (WT) controls. These findings suggest a regulatory crosstalk between autophagy and axonal degeneration pathways, which is mediated through ULK1-SARM1 interaction and contributes to the ability of SARM1 to accumulate in injured axons.

Background: Immune-checkpoint inhibitors (ICIs) changed the therapeutic landscape of patients with lung cancer. However, only a subset of them derived clinical benefit and evidenced the need to identify reliable predictive biomarkers. Liquid biopsy is the non-invasive and repeatable analysis of biological material in body fluids and a promising tool for cancer biomarkers discovery. In particular, there is growing evidence that extracellular vesicles (EVs) play an important role in tumor progression and in tumor-immune interactions. Thus, we evaluated whether extracellular vesicle PD-L1 expression could be used as a biomarker for prediction of durable treatment response and survival in patients with non-small cell lung cancer (NSCLC) undergoing treatment with ICIs., Methods: Dynamic changes in EV PD-L1 were analyzed in plasma samples collected before and at 9 ± 1 weeks during treatment in a retrospective and a prospective independent cohorts of 33 and 39 patients, respectively., Results: As a result, an increase in EV PD-L1 was observed in non-responders in comparison to responders and was an independent biomarker for shorter progression-free survival and overall survival. To the contrary, tissue PD-L1 expression, the commonly used biomarker, was not predictive neither for durable response nor survival., Conclusion: These findings indicate that EV PD-L1 dynamics could be used to stratify patients with advanced NSCLC who would experience durable benefit from ICIs., (© 2022. The Author(s).)

We previously observed that the nine-member family of autotransported polymorphic membrane proteins (Pmps) of Chlamydia trachomatis is variably expressed in cell culture. Additionally, C. trachomatis -infected patients display variable Pmp-specific serum antibody profiles indirectly suggesting expression of unique Pmp profiles is an adaptive response to host-specific stimuli during infection. Here, we propose that the host response to Pmps and other outer surface proteins may correlate with disease severity. This study tests this hypothesis using an ELISA that measures serum IgG antibodies specific for the nine C. trachomatis Pmp subtypes and four immunodominant antigens (MOMP, OmcB, Hsp60, ClpP) in 265 participants of the Chlamydia Adolescent/Young Adult Reproductive Management (CHARM) cohort. More C. trachomatis -infected females displayed high Pmp-specific antibody levels (cut-off Indexes) than males (35.9%-40.7% of females vs . 24.2%-30.0% of males), with statistical significance for PmpC, F and H ( P < 0.05). Differences in Pmp-specific antibody profiles were not observed between C. trachomatis -infected females with a clinical diagnosis of pelvic inflammatory disease (PID) and those without. However, a statistically significant association between high levels of OmcB-specific antibody and a PID diagnosis ( P < 0.05) was observed. Using antibody levels as an indirect measure of antigen expression, our results suggest that gender- and/or site-specific (cervix in females vs . urethra in males) stimuli may control pmp expression in infected patients. They also support the possible existence of immune biomarkers of chlamydial infection associated with disease and underline the need for high resolution screening in human serum., Competing Interests: None declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Mechanical forces are conducted through myofibers and into nuclei to regulate muscle development, hypertrophy, and homeostasis. We hypothesized that nuclei in aged muscle have changes in the nuclear envelope and associated proteins, resulting in altered markers of mechano-signaling., Methods: YAP/TAZ protein expression and gene expression of downstream targets, Ankrd1 and Cyr61, were evaluated as mechanotransduction indicators. Expression of proteins in the nuclear lamina and the nuclear pore complex (NPC) were assessed, and nuclear morphology was characterized by electron microscopy. Nuclear envelope permeability was assessed by uptake of 70 kDa fluorescent dextran., Results: Nuclear changes with aging included a relative decrease of lamin β1 and Nup107, and a relative increase in Nup93, which could underlie the aberrant nuclear morphology, increased nuclear leakiness, and elevated YAP/TAZ signaling., Conclusion: Aged muscles have hyperactive nuclear-cytoplasmic signaling, indicative of altered nuclear mechanotransduction. These data highlight a possible role for the nucleus in aging-related aberrant mechano-sensing., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The family Chlamydiaceae currently comprises a single genus Chlamydia, with 11 validly published species and seven more taxa. It includes the human pathogens Chlamydia (C.) trachomatis, C. pneumoniae and C. psittaci, a zoonotic agent causing avian chlamydiosis and human psittacosis, as well as other proven or potential pathogens in ruminants, birds, snakes, reptiles and turtles. During routine testing of 15 apparently healthy captive flamingos in a zoo in 2011, an atypical strain of Chlamydiaceae was detected by real-time PCR of cloacal swab samples. Sequence analysis of the 16S rRNA gene revealed high similarity to the uncultured Chlamydiales bacterium clone 122, which previously had been found in gulls. As more samples were collected during annual campaigns of the flamingo ringing program in southern France from 2012 to 2015, Chlamydiaceae-specific DNA was detected by PCR in 30.9% of wild birds. From these samples, three strains were successfully grown in cell culture. Ultrastructural analysis, comparison of 16S and 23S rRNA gene sequences, whole-genome analysis based on de novo hybrid-assembled sequences of the new strains as well as subsequent calculation of taxonomic parameters revealed that the relatedness of the flamingo isolates to established members of the family Chlamydiaceae was sufficiently distant to indicate that the three strains belong to two distinct species within a new genus. Based on these data, we propose the introduction of Chlamydiifrater gen. nov., as a new genus, and Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov., as two new species of the genus., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Pancreatic cancer represents a life threatening disease with rising mortality. Although the synergistic combination of gemcitabine and albumin-bound paclitaxel has proven to enhance the median survival rates as compared to gemcitabine alone, their systemic and repeated co-administration has been associated with serious toxic side effects and poor patient compliance. For this purpose, we designed a thermosensitive and biodegradable hydrogel encapsulating targeted nanoparticles for the local and sustained delivery of gemcitabine (GEM) and paclitaxel (PTX) to pancreatic cancer. GEM and PTX were loaded into PR&#95;b-functionalized liposomes targeting integrin α 5 β 1 , which was shown to be overexpressed in pancreatic cancer. PR&#95;b is a fibronectin-mimetic peptide that binds to α 5 β 1 with high affinity and specificity. The PR&#95;b liposomes were encapsulated into a poly(δ-valerolactone-co-D,L-lactide)-b-poly(ethylene glycol)-b-poly(δ-valerolactone-co-D,L-lactide) (PVLA-PEG-PVLA) hydrogel and demonstrated sustained release of both drugs compared to PR&#95;b-functionalized liposomes free in solution or free drugs in the hydrogel. Moreover, the hydrogel-nanoparticle system was proven to be very efficient towards killing monolayers of human pancreatic cancer cells (PANC-1), and showed a significant reduction in the growth pattern of PANC-1 tumor spheroids as compared to hydrogels encapsulating non-targeted liposomes with GEM/PTX or free drugs, after a one week treatment period. Our hybrid hydrogel-nanoparticle system is a promising platform for the local and sustained delivery of GEM/PTX to pancreatic cancer, with the goal of maximizing the therapeutic efficacy of this synergistic drug cocktail while potentially minimizing toxic side effects and eliminating the need for repeated co-administration., (Copyright © 2020 Elsevier B.V. All rights reserved.)

"Giant" phages have genomes of >200 kbp, confined in correspondingly large capsids whose assembly and maturation are still poorly understood. Nevertheless, the first assembly product is likely to be, as in other tailed phages, a procapsid that subsequently matures and packages the DNA. The associated transformations include the cleavage of many proteins by the phage-encoded protease, as well as the thinning and angularization of the capsid. We exploited an amber mutation in the viral protease gene of the Salmonella giant phage SPN3US, which leads to the accumulation of a population of capsids with distinctive properties. Cryo-electron micrographs reveal patterns of internal density different from those of the DNA-filled heads of virions, leading us to call them "mottled capsids". Reconstructions show an outer shell with T = 27 symmetry, an embellishment of the HK97 prototype composed of the major capsid protein, gp75, which is similar to some other giant viruses. The mottled capsid has a T = 1 inner icosahedral shell that is a complex network of loosely connected densities composed mainly of the ejection proteins gp53 and gp54. Segmentation of this inner shell indicated that a number of densities (~12 per asymmetric unit) adopt a "twisted hook" conformation. Large patches of a proteinaceous tetragonal lattice with a 67 Å repeat were also present in the cell lysate. The unexpected nature of these novel inner shell and lattice structures poses questions as to their functions in virion assembly.

The head of Salmonella virus SPN3US is composed of ~50 different proteins and is unusual because within its packaged genome there is a mass (>40 MDa) of ejection or E proteins that enter the Salmonella cell. The assembly mechanisms of this complex structure are poorly understood. Previous studies showed that eight proteins in the mature SPN3US head had been cleaved by the prohead protease. In this study, we present the characterization of SPN3US prohead protease mutants using transmission electron microscopy and mass spectrometry. In the absence of the prohead protease, SPN3US head formation was severely impeded and proheads accumulated on the Salmonella inner membrane. This impediment is indicative of proteolysis being necessary for the release and subsequent DNA packaging of proheads in the wild-type phage. Proteomic analyses of gp245- proheads that the normal proteolytic processing of head proteins had not occurred. Assays of a recombinant, truncated form of the protease found it was active, leading us to hypothesize that the C -terminal propeptide has a role in targeting the protease into the prohead core. Our findings provide new evidence regarding the essential role of proteolysis for correct head assembly in this remarkable parasite.

The commercial marketplace has seen a rapid increase in the number of over-the-counter charcoal-containing mouthwashes. The purpose of this systemic review was to examine the clinical and laboratory evidence supporting therapeutic claims of efficacy and safety of use of charcoal-based mouthwashes. Secondly, the product labels and information of 36 commercially marketed charcoal mouthwashes were reviewed for active ingredients. Only 8% of charcoal mouthwashes contained an active ingredient, such as cetylpyridinium chloride or chlorhexidine. There is insufficient evidence to substantiate the therapeutic and cosmetic marketing claims of charcoal-based mouthwashes, including antimicrobial activity, anti-halitosis, tooth whitening, periodontal disease control, caries reduction and tooth remineralisation, among others. Moreover, there is no available information on charcoal particulate size or abrasivity of any of these products. Dental clinicians should advise their patients to exercise caution when using over-the-counter charcoal-containing mouthwashes because of the lack of evidence supporting therapeutic or cosmetic effectiveness as well as safety.

Chlamydiaceae are obligate intracellular bacterial pathogens for humans and animals. A recent study highlighted that a Chlamydiaceae intermediary between C. psittaci and C. abortus can infect hawks. Here, an isolate was obtained upon passage of cloacal and conjunctival sac material collected from a female hatch-year red-shouldered hawk (Buteo lineatus) in cultured cells. The diseased bird, one of 12 birds housed in a rehabilitation center, developed conjunctivitis and later died. Swabs from both sites tested positive for Chlamydia using the QuickVue Chlamydia test. The isolate, named RSHA, tested negative in qPCR assays specific for C. psittaci and C. abortus, respectively. Analysis of the 16S rRNA, 23S rRNA and whole genome sequences as well as MLST, ANIb and TETRA values reveal that C. psittaci and C. abortus are the closest relatives of RSHA. However, the overall results strongly suggest a phylogenetic intermediate position between these two species. Therefore, we propose the introduction of a new species designated Chlamydia buteonis with RSHA T as the type strain., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Many intracellular pathogens subvert host membrane trafficking pathways to promote their replication. Toxoplasma multiplies in a membrane-bound parasitophorous vacuole (PV) that interacts with mammalian host organelles and intercepts Golgi Rab vesicles to acquire sphingolipids. The mechanisms of host vesicle internalization and processing within the PV remain undefined. We demonstrate that Toxoplasma sequesters a broad range of Rab vesicles into the PV. Correlative light and electron microscopy analysis of infected cells illustrates that intravacuolar Rab1A vesicles are surrounded by the PV membrane, suggesting a phagocytic-like process for vesicle engulfment. Rab11A vesicles concentrate to an intravacuolar network (IVN), but this is reduced in Δgra2 and Δgra2Δgra6 parasites, suggesting that tubules stabilized by the TgGRA2 and TgGRA6 proteins secreted by the parasite within the PV contribute to host vesicle sequestration. Overexpression of a phospholipase TgLCAT, which is localized to the IVN, results in a decrease in the number of intravacuolar GFP-Rab11A vesicles, suggesting that TgLCAT controls lipolytic degradation of Rab vesicles for cargo release., (© 2017 Romano et al.)

This article presents the results of a scanning electron microscope (SEM) analysis of the surface of an acrylic custom provisional abutment following first disconnection from a post-extraction immediate implant placement. An implant was placed immediately after extraction, the site was grafted, and a barrier membrane was adapted for graft containment. A custom acrylic shell was then relined, polished, and steam-cleaned prior to being screwed onto the implant. After 5 months of undisturbed healing, the custom provisional abutment was disconnected for the first time and processed for SEM examination. The surface of the custom acrylic abutment revealed well-spread fibroblast-like cells with filopodia inserting into the porous surface. These observations suggest that the surface topography of the acrylic provisional restoration/ abutment can function as a substratum for cellular adhesion and may serve an important role in supporting peri-implant mucosa at the time of immediate implant placement.

The polymorphic membrane protein (Pmp) paralogous families of Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia abortus are putative targets for Chlamydia vaccine development. To determine whether this is also the case for Pmp family members of C. psittaci, we analyzed transcription levels, protein production and localization of several Pmps of C. psittaci. Pmp expression profiles were characterized using quantitative real-time PCR (RT-qPCR), immunofluorescence (IF) and immuno-electron microscopy (IEM) under normal and stress conditions. We found that PmpA was highly produced in all inclusions as early as 12 hpi in all biological replicates. In addition, PmpA and PmpH appeared to be unusually accessible to antibody as determined by both immunofluorescence and immuno-electron microscopy. Our results suggest an important role for these Pmps in the pathogenesis of C. psittaci, and make them promising candidates in vaccine development., Competing Interests: The authors have declared that no competing interests exist.

SINC, a new type III secreted protein of the avian and human pathogen Chlamydia psittaci, uniquely targets the nuclear envelope of C. psittaci-infected cells and uninfected neighboring cells. Digitonin-permeabilization studies of SINC-GFP-transfected HeLa cells indicate that SINC targets the inner nuclear membrane. SINC localization at the nuclear envelope was blocked by importazole, confirming SINC import into the nucleus. Candidate partners were identified by proximity to biotin ligase-fused SINC in HEK293 cells and mass spectrometry (BioID). This strategy identified 22 candidates with high confidence, including the nucleoporin ELYS, lamin B1, and four proteins (emerin, MAN1, LAP1, and LBR) of the inner nuclear membrane, suggesting that SINC interacts with host proteins that control nuclear structure, signaling, chromatin organization, and gene silencing. GFP-SINC association with the native LEM-domain protein emerin, a conserved component of nuclear "lamina" structure, or with a complex containing emerin was confirmed by GFP pull down. Our findings identify SINC as a novel bacterial protein that targets the nuclear envelope with the capability of globally altering nuclear envelope functions in the infected host cell and neighboring uninfected cells. These properties may contribute to the aggressive virulence of C. psittaci., (© 2015 Mojica et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Introduction: We adopted a proteomics-based approach to gain insights into phenotypic differences between A/J and B10.SJL murine dysferlinopathy models., Methods: We optimized immunoblotting of dysferlin by preparing homogenates of the tibialis anterior (TA) muscle under several different conditions. We compared TA muscles of control, A/J, and B10.SJL mice for levels of dysferlin; dysferlin's partners MG53, annexin-A2, and caveolin-3; and the endoplasmic reticulum (ER) stress marker CHOP. We performed immunoelectron microscopy on control rat TA muscle to determine the precise location of dysferlin., Results: RIPA (radioimmunoprecipitation assay) buffer and sonication improves immunoblotting of dysferlin. The ER stress marker CHOP is elevated in A/J muscle. Dysferlin is localized mostly to membranes close to the Z-disk that have been reported to be part of the Golgi, ER, and sarcoplasmic reticulum (SR) networks., Conclusions: ER stress might underlie phenotypic differences between A/J and B10.SJL mice and play a role in human dysferlinopathies., (Copyright © 2014 Wiley Periodicals, Inc.)

Among chlamydial virulence factors are the type III secretion (T3S) system and its effectors. T3S effectors target host proteins to benefit the infecting chlamydiae. The assortment of effectors, each with a unique function, varies between species. This variation likely contributes to differences in host specificity and disease severity. A dozen effectors of Chlamydia trachomatis have been identified; however, estimates suggest that more exist. A T3S prediction algorithm, SVM-based Identification and Evaluation of Virulence Effectors (SIEVE), along with a Yersinia surrogate secretion system helped to identify a new T3S substrate, CT082, which rather than functioning as an effector associates with the chlamydial envelope after secretion. SIEVE was modified to improve/expand effector predictions to include all sequenced genomes. Additional adjustments were made to the existing surrogate system whereby the N terminus of putative effectors was fused to a known effector lacking its own N terminus and was tested for secretion. Expansion of effector predictions by cSIEVE and modification of the surrogate system have also assisted in identifying a new T3S substrate from C. psittaci. The expanded predictions along with modifications to improve the surrogate secretion system have enhanced our ability to identify novel species-specific effectors, which upon characterization should provide insight into the unique pathogenic properties of each species., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Investigations conducted on feral African Sacred Ibises (Threskiornisaethiopicus) in western France led to the isolation of a strain with chlamydial genetic determinants. Ultrastructural analysis, comparative sequence analysis of the 16S rRNA gene, ompA, and of a concatenate of 31 highly conserved genes, as well as determination of the whole genome sequence confirmed the relatedness of the new isolate to members of the Chlamydiaceae, while, at the same time demonstrating a unique position outside the currently recognized species of this family. We propose to name this new chlamydial species Chlamydiaibidis .

The Nannochloropsis genus contains oleaginous microalgae that have served as model systems for developing renewable biodiesel. Recent genomic and transcriptomic studies on Nannochloropsis species have provided insights into the regulation of lipid production in response to nitrogen stress. Previous studies have focused on the responses of Nannochloropsis species to short-term nitrogen stress, but the effect of long-term nitrogen deprivation remains largely unknown. In this study, physiological and proteomic approaches were combined to understand the mechanisms by which Nannochloropsis oceanica IMET1 is able to endure long-term nitrate deprivation and its ability to recover homeostasis when nitrogen is amended. Changes of the proteome during chronic nitrogen starvation espoused the physiological changes observed, and there was a general trend toward recycling nitrogen and storage of lipids. This was evidenced by a global down-regulation of protein expression, a retained expression of proteins involved in glycolysis and the synthesis of fatty acids, as well as an up-regulation of enzymes used in nitrogen scavenging and protein turnover. Also, lipid accumulation and autophagy of plastids may play a key role in maintaining cell vitality. Following the addition of nitrogen, there were proteomic changes and metabolic changes observed within 24 h, which resulted in a return of the culture to steady state within 4 d. These results demonstrate the ability of N. oceanica IMET1 to recover from long periods of nitrate deprivation without apparent detriment to the culture and provide proteomic markers for genetic modification.

Secondary caries remains the main reason for dental restoration failure. Replacement of failed restorations accounts for 50%-70% of all restorations performed. Antibacterial adhesives could inhibit biofilm acids at tooth-restoration margins, and calcium phosphate (CaP) ions could remineralize tooth lesions. The objectives of this study were to: (1) incorporate nanoparticles of silver (NAg), quaternary ammonium dimethacrylate (QADM), and nanoparticles of amorphous calcium phosphate (NACP) into bonding agent; and (2) investigate their effects on dentin bonding and microcosm biofilms. An experimental primer was made with pyromellitic glycerol dimethacrylate (PMGDM) and 2-hydroxyethyl methacrylate (HEMA). An adhesive was made with bisphenol-A-glycerolate dimethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA). NAg was incorporated into primer at 0.1 wt %. The adhesive contained 0.1% NAg and 10% QADM, and 0%-40% NACP. Incorporating NAg into primer and NAg-QADM-NACP into adhesive did not adversely affect dentin bond strength (p > 0.1). Scanning electron microscopy showed numerous resin tags, and transmission electron microscopy revealed NAg and NACP in dentinal tubules. Viability of human saliva microcosm biofilms on primer/adhesive/composite disks was substantially reduced via NAg and QADM. Metabolic activity, lactic acid, and colony-forming units of biofilms were much lower on the new bonding agents than control (p < 0.05). In conclusion, novel dental bonding agents containing NAg, QADM, and NACP were developed with the potential to kill residual bacteria in the tooth cavity and inhibit the invading bacteria along tooth-restoration margins, with NACP to remineralize tooth lesions. The novel method of combining antibacterial agents (NAg and QADM) with remineralizing agent (NACP) may have wide applicability to other adhesives for caries inhibition., (Copyright © 2012 Wiley Periodicals, Inc.)

Cellular energy homeostasis is a crucial function of oxidative tissues but becomes altered with obesity, a major health problem that is rising unabated and demands attention. Maintaining cardiac lipid homeostasis relies on complex processes and pathways that require concerted actions between lipid droplets (LDs) and mitochondria to prevent intracellular accumulation of bioactive or toxic lipids while providing an efficient supply of lipid for conversion into ATP. While cardiac mitochondria have been extensively studied, cardiac LDs and their role in heart function have not been fully characterized. The cardiac LD compartment is highly dynamic and individual LD is small, making their study challenging. Here, we describe a simple procedure to isolate cardiac LDs that provide sufficient amounts of highly enriched material to allow subsequent protein and lipid biochemical characterization. We also present a detailed protocol to image cardiac LDs by conventional transmission electronic microscopy to provide two-dimensional (2D) analyses of cardiac LDs and mitochondria. Finally, we discuss the potential advantages of dual ion beam and electron beam platform (FIB-SEM) technology to study the cardiac LDs and mitochondria by allowing 3D imaging analysis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Late Chlamydia trachomatis inclusions express each member of the surface-exposed polymorphic membrane protein family (Pmp subtypes A through I) with a reproducible distribution of fully-on, fully-off and intermediate phenotypes. This observation is consistent with observed variable Pmp antibody profiles in C. trachomatis-infected patients and has led to the hypothesis that the pmp gene family forms the basis of a phase variation-like mechanism of antigenic variation. Here we investigate and compare the developmental expression of each of the nine pmp genes under conditions of optimal in vitro growth with that under conditions that promote prolonged survival of chlamydiae when exposed to penicillin-induced stress. We demonstrate that the pmp gene family includes distinct transcriptional units that are differentially expressed along development and differentially responsive to stress. In particular, our results indicate that expression of pmpA, pmpD and pmpI is uniquely unaffected by stress, suggesting that the PmpA, PmpD and PmpI proteins play a critical role in the pathogenesis of C. trachomatis., (© 2011 Blackwell Publishing Ltd.)

Although endosomal compartments have been suggested to play a role in unconventional protein secretion, there is scarce experimental evidence for such involvement. Here we report that recycling endosomes are essential for externalization of cytoplasmic secretory protein tissue transglutaminase (tTG). The de novo synthesized cytoplasmic tTG does not follow the classical ER/Golgi-dependent secretion pathway, but is targeted to perinuclear recycling endosomes, and is delivered inside these vesicles prior to externalization. On its route to the cell surface tTG interacts with internalized β1 integrins inside the recycling endosomes and is secreted as a complex with recycled β1 integrins. Inactivation of recycling endosomes, blocking endosome fusion with the plasma membrane, or downregulation of Rab11 GTPase that controls outbound trafficking of perinuclear recycling endosomes, all abrogate tTG secretion. The initial recruitment of cytoplasmic tTG to recycling endosomes and subsequent externalization depend on its binding to phosphoinositides on endosomal membranes. These findings begin to unravel the unconventional mechanism of tTG secretion which utilizes the long loop of endosomal recycling pathway and indicate involvement of endosomal trafficking in non-classical protein secretion.

The hypothesized variable expression of polymorphic membrane proteins (PmpA-PmpI) in Chlamydia trachomatis-infected patients was tested by examination of the expression of each Pmp subtype in in vitro-grown C. trachomatis. A panel of monospecific polyclonal and monoclonal antibodies was used to demonstrate surface exposure of Pmps of each subtype by differential immunofluorescence (IF) with and without prior detergent permeabilization of paraformaldehyde-fixed inclusions and for selected Pmps by immunogold labelling. Although specific transcript was detected for each pmp gene late in development, IF experiments with Pmp subtype-specific antibodies reveal that a number of inclusions in a single infection do not express Pmps of a given subtype. Coexpression experiments suggest that pmp genes are shut off independently from one another in non-expressing inclusions, i.e. different inclusions are switched off for different Pmps. Overall, these studies establish the existence of an efficient shutoff mechanism independently affecting the expression of each member of the pmp gene family in in vitro-grown C. trachomatis. Like other paralogous gene families of bacterial pathogens, the pmp gene family of C. trachomatis may serve the critical dual function of a highly adaptable virulence factor also providing antigenic diversity in the face of the host adaptive immune response.

Genomic analysis of the Chlamydiaceae has revealed a multigene family encoding large, putatively autotransported polymorphic membrane proteins (Pmps) with nine members in the sexually transmitted pathogen Chlamydia trachomatis. While various pathogenesis-related functions are emerging for the Pmps, observed genotypic and phenotypic variation among several chlamydial Pmps in various Chlamydia species has led us to hypothesize that the pmp gene repertoire is the basis of a previously undetected mechanism of antigenic variation. To test this hypothesis, we chose to examine the serologic response of C. trachomatis-infected patients to each Pmp subtype. Immune serum samples were collected from four populations of patients with confirmed C. trachomatis genital infection: 40 women with pelvic inflammatory disease from Pittsburgh, PA; 27 and 34 adolescent/young females from Oakland, CA, and Little Rock, AR, respectively; and 58 adult male patients from Baltimore, MD. The Pmp-specific antibody response was obtained using immunoblot analysis against each of the nine recombinantly expressed Pmps and quantified by densitometry. Our results show that nearly all C. trachomatis-infected patients mount a strong serologic response against individual or multiple Pmp subtypes and that the antibody specificity profile varies between patients. Moreover, our analysis reveals differences in the strengths and specificities of the Pmp subtype-specific antibody reactivity relating to gender and clinical outcome. Overall, our results indicate that the Pmps elicit various serologic responses in C. trachomatis-infected patients and are consistent with the pmp gene family being the basis of a mechanism of antigenic variation.

We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals. Two high throughput approaches were used. First, an in silico screening identified 200 conserved putative B. anthracis spore components. A total of 192 of those candidate genes were expressed and purified in vitro, 75 of which reacted with the rabbit immune sera generated against B. anthracis spores. The second approach was to screen for cross-reacting antigens in the spore proteome of 10 diverse B. cereus group strains. Two-dimensional electrophoresis resolved more than 200 protein spots in each spore preparation. About 72% of the protein spots were found in all the strains. 18 of these conserved proteins reacted against anti-B. anthracis spore rabbit immune sera, two of which (alanine racemase, Dal-1 and the methionine transporter, MetN) overlapped the set of proteins identified using the in silico screen. A conserved repeat domain protein (Crd) was the most immunoreactive protein found broadly across B. cereus sensu lato strains. We have established an approach for finding conserved targets across a species using population genomics and proteomics. The results of these screens suggest the possibility of a multiepitope antigen for broad host range diagnostics or therapeutics against Bacillus spore infection.

Infections caused by the obligate intracellular pathogen Chlamydia trachomatis have a marked impact on human health. C. trachomatis serovariants are the leading cause of bacterial sexually transmitted disease and infectious preventable blindness. Despite decades of effort, there is no practical vaccine against C. trachomatis diseases. Here we report that all C. trachomatis reference serotypes responsible for sexually transmitted disease and blinding trachoma synthesize a highly conserved surface-exposed antigen termed polymorphic membrane protein D (PmpD). We show that Ab specific to PmpD are neutralizing in vitro. We also present evidence that Ab against serovariable-neutralizing targets, such as the major outer membrane protein, block PmpD neutralization. This finding suggests that a decoy-like immune evasion strategy may be active in vivo whereby immunodominant type-specific surface antigens block the neutralizing ability of species-common PmpD Ab. Collectively, these results show that PmpD is a previously uncharacterized C. trachomatis species-common pan-neutralizing target. Moreover, a vaccine protocol using recombinant PmpD to elicit neutralizing Ab in the absence of immunodominant type-specific Ab might be highly efficacious and surpass the level of protection achieved through natural immunity.

Chlamydia trachomatis is an intracellular bacterium that causes ocular and urogenital diseases worldwide. Membrane proteins have only been partially characterized, and the discovery of a nine-member polymorphic membrane protein gene family has enhanced interest in defining their function. We previously reported two putative insertion sequence-like elements in pmpC for biovariant Ba and one each for G and L2, suggesting horizontal gene transfer. Because of this and the tissue tropism differences for these biovariants, we analyzed by quantitative real-time RT-PCR pmpC expression relative to immunogenic protein genes ompA, groEL and gseA throughout development. Sera from infected adolescents were reacted by immunoblot against recombinant (r)PmpC and rMOMP. ompA and groEL revealed different developmental transcriptome profiles among the biovariants. pmpC expression occurred at 2 h, peaked at 18 for L2 (at 24 for Ba and G), with the highest mRNA levels throughout development for L2. pmpC expression as a function of time paralleled ompA expression with higher mRNA levels compared with groEL later in development. Only sera from D-, E- and G-infected patients reacted to rPmpC; all infected patients reacted to rMOMP. pmpC expression during logarithmic growth suggests a role in membrane building and/or integrity, which is supported by the presence of a signal peptidase and C-terminal phenylalanine in PmpC. Because phylogenetic analyses of pmpC segregate serovars according to tissue tropism, we speculate that biovariant transcriptome differences may contribute to this tropism. The heterogeneous biovariant pmpC expression throughout development and differential PmpC immunoreactivity also suggest a role for pmpC in antigenic variation.

The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.

The authors report the isolation and molecular characterization of a bacteriophage, &phi;CPG1, which infects CHLAMYDIA: psittaci strain Guinea pig Inclusion Conjunctivitis. Purified virion preparations contained isometric particles of 25 nm diameter, superficially similar to spike-less members of the &phi;X174 family of bacteriophages. The single-stranded circular DNA genome of &phi;CPG1 included five large ORFs, which were similar to ORFs in the genome of a previously described CHLAMYDIA: bacteriophage (Chp1) that infects avian C. psittaci. Three of the ORFs encoded polypeptides that were similar to those in a phage infecting the mollicute Spiroplasma melliferum, a pathogen of honeybees. Lesser sequence similarities were seen between two ORF products and the major capsid protein of the &phi;X174 coliphage family and proteins mediating rolling circle replication initiation in phages, phagemids and plasmids. Phage &phi;CPG1 is the second member of the genus CHLAMYDIAMICROVIRUS:, the first to infect a member of a CHLAMYDIA: species infecting mammals. Similarity searches of the nucleotide sequence further revealed a highly conserved (75% identity) 375 base sequence integrated into the genome of the human pathogen Chlamydia pneumoniae. This genomic segment encodes a truncated 113 residue polypeptide, the sequence of which is 72% identical to the amino-terminal end of the putative replication initiation protein of &phi;CPG1. This finding suggests that C. pneumoniae has been infected by a phage related to &phi;CPG1 and that infection resulted in integration of some of the phage genome into the C. pneumoniae genome.

Three recently discovered ssDNA Chlamydia-infecting microviruses, phiCPG1, phiAR39, and Chp2, were compared with the previously characterized phage from avian C. psittaci, Chp1. Although the four bacteriophages share an identical arrangement of their five main genes, Chpl has diverged significantly in its nucleotide and protein sequences from the other three, which form a closely related group. The VP1 major viral capsid proteins of phiCPG1 and phiAR39 (from guinea pig-infecting C. psittaci and C. pneumoniae, respectively) are almost identical. However, VP1 of ovine C. psittaci phage Chp2 shows a high rate of nucleotide sequence change localized to a region encoding the "IN5" loop of the protein, thought to be a potential receptor-binding site. Phylogenetic analysis suggests that the ORF4 replication initiation protein is evolving faster than the other phage proteins. phiCPG1, phiAR39, and Chp2 are closely related to an ORF4 homolog inserted in the C. pneumoniae chromosome. This sequence analysis opens the way toward understanding the host-range and evolutionary history of these phages.

Four genes of Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), whose predicted products are highly homologous to structural and regulatory components of a contact-dependent or type III secretion apparatus, were isolated. Related to genes present in several animal and plant bacterial pathogens, these genes may represent a section of a previously undetected chromosomal virulence locus analogous to several recently described virulence-associated type III secretion loci. The existence of contact-dependent secretion in Chlamydia strongly suggests that these bacteria use pathogenic mechanisms that are similar to those of other intracellular bacterial pathogens. Unlike other intracellular bacteria, however, chlamydiae are metabolically inactive extracellularly and only become capable of global protein synthesis several hours after infection. This implies that chlamydial contact-dependent secretion is only active from within, uniquely after the bacteria have been internalized by eukaryotic cells. The possible role(s) of this pathway in chlamydial pathogenesis are discussed.

The nucleotide sequence of nearly 6 kb of genomic DNA located immediately upstream of the omp3-omp2 operon of Chlamydia psittaci strain GPIC was obtained, revealing four significant open reading frames (ORFs), named ORF1, ORF2, ORF4 and ORF5. Searches for homologous sequences in the GenBank/EMBL databases have revealed that: (a) the open-ended ORF1 putatively encodes an homolog of RecJ of Escherichia coli, thought to be required for RecBCD-independent and conjugational recombination, and for UV repair; (b) the predicted translation product of ORF4 is highly homologous to the putative product of EUO, a previously described ORF of avian C. psittaci strain 6BC which is preferentially transcribed early during the life cycle; and (c) ORF5 putatively encodes an homolog of bacterial glutamyl-tRNA synthetases. This analysis establishes the genetic linkage of late (omp3-omp2) and of a proposed early (EUO) genes in Chlamydia.

The nucleotide sequence of a 3.1-kb genomic DNA fragment carrying the omp3, omp2 and srp gene homologs from Chlamydia psittaci strain GPIC was determined. A comparative analysis of the GPIC sequence with other chlamydial omp2-linked sequences reveals highly conserved omp3 and omp2 upstream sequences across species, suggesting a unified mechanism of transcription regulation. In contrast, the omp2-srp intergenic segment, which encompasses hypothetical srp transcriptional initiation sites, is relatively less conserved in length and in sequence. Examination of the predicted translation products reveals a high degree of homology within Omp3 and Omp2 across species, with the notable exception of the N-terminal fifth of Omp2. Although the latter segment displays relatively high interspecies sequence variation, it includes a smaller segment, whose high positive charge density is conserved across species, suggesting a conserved structure/function. In contrast to Omp2 and Omp3, a comparative analysis of the predicted amino acid (aa) sequence of the srp product reveals high homology within species, but relatively little across species. A 38-aa segment near the C-terminus of Srp, whose sequence is 64% identical between C. psittaci GPIC and C. trachomatis, is partially truncated in C. psittaci 6BC.

The chlamydial life cycle involves the intimate interaction of components of the infectious elementary body (EB) surface with receptors on the susceptible eukaryotic cell plasma membrane. We have developed an in vitro ligand binding assay system for the identification and characterization of detergent-extracted EB envelope proteins capable of binding to glutaraldehyde-fixed HeLa cell surfaces. With this assay, the developmentally regulated cysteine-rich envelope protein Omp2 of Chlamydia psittaci strain guinea pig inclusion conjunctivitis was shown to bind specifically to HeLa cells. HeLa cells bound Omp2 selectively over other cell wall-associated proteins, including the major outer membrane protein, and the binding of Omp2 was abolished under conditions which alter its conformation. Furthermore, trypsin treatment, which reduces EB adherence, resulted in the proteolytic removal of a small terminal peptide of Omp2 at the EB surface and inactivated Omp2 in the ligand binding assay, while having a negligible effect on the major outer membrane protein. Collectively, our results suggest that Omp2 possesses the capacity to engage in a specific interaction with the host eukaryotic cell. We speculate that, since Omp2 is present only in the infectious EB form, the observed in vitro interaction may be representative of a determining step of the chlamydial pathogenic process.

The outcome of infection is determined by both the quantity and the quality of an induced immune response. In particular, it has been demonstrated for selected pathogens that induction of TH1 or TH2 type helper T-cell subsets determines whether an immune response gives rise to protective immunity or disease-associated immunopathology. The nature of the antigen and the type of antigen-presenting cells recruited in the induction of a response are critical factors that influence the quality of the immune response. Of particular interest in this respect is the immune response to bacterial particles and the impact of cell wall-associated lipopolysaccharide (LPS) on that response. Nonspecific activation of macrophages and B lymphocytes by LPS could skew the phenotype of activated antigen-presenting cells and selectively alter the immunoglobulin isotypes and helper T-cell subsets that are induced following infection. In an initial attempt to detect immune deviation associated with LPS stimulation, we have compared the immunoglobulin isotypes of antibodies specific for the cysteine-rich outer membrane protein Omp2 induced in normal and LPS-hyporesponsive mice following immunization with Chlamydia psittaci strain guinea pig inclusion conjunctivitis whole elementary bodies. We report that there is a dramatic shift of Omp2-specific antibody from predominantly immunoglobulin G2a (IgG2a) isotype in LPS-hyporesponsive mice to high levels of IgG1 isotype in LPS-responder strains. The dependence of the IgG1 isotype shift on the LPS responder status is linked to the structure of the antigen and its natural processing pathway since LPS-hyporesponsive mice are not, in general, deficient in IgG1 antibody production. In particular, the antibody response to purified recombinant Omp2 is predominantly of the IgG1 isotype even in LPS-hyporesponsive mice.

Chlamydia trachomatis is an important human pathogen. Research to develop a Chlamydia vaccine has focused on the major outer membrane protein (MOMP). Determinants of this protein elicit serovar-specific neutralizing antibodies which are thought to play a critical role in protective immunity. MOMP-specific antibody responses are highly variable in the polymorphic population. Genetic factors which might influence the MOMP-specific immune response are consequently of particular interest. The C. psittaci strain guinea pig inclusion conjunctivitis (GPIC) is a natural pathogen of the guinea pig that causes both ocular and genital tract infections that closely resemble those caused by C. trachomatis in humans. As such, it provides an excellent model for disease. In this report, we explore the influence of major histocompatibility complex-linked genes on the MOMP-specific antibody response in mice immunized with either whole GPIC elementary bodies or recombinant GPIC MOMP. Our results indicate that the MOMP-specific antibody response is major histocompatibility complex linked such that mice of the H-2d haplotype are high responders while mice of the H-2k haplotype are low responders. We demonstrate that MOMP-specific B cells are present in H-2k strains which are, however, deficient in MOMP-specific helper T cells. Although immunization of low-MOMP-responder strains with whole chlamydial elementary bodies induces high levels of immunoglobulin G antibody specific for Omp2, the cysteine-rich outer membrane protein, MOMP-specific B cells are unable to receive help from Omp2-specific T cells. The failure of intermolecular help from Omp2-specific T cells and related observations raise important issues regarding the processing and presentation of chlamydial antigens and the design of optimal subunit vaccines.

While enteroinvasive Escherichia coli (EIEC) and shigellae are genotypically nearly identical, a difference has been reported in the infective dose to humans: EIEC is 10,000-fold less infectious than shigellae. A possible basis for this difference lies in the inherent invasiveness of these bacteria toward epithelial cells. Thus, despite the high degree of homology between the invasion plasmids of EIEC and shigellae, substantial differences in genetic organization and/or sequence may exist. We have undertaken a systematic genetic analysis of the EIEC plasmid pSF204, using transposon mutagenesis. Congo red-negative TnphoA insertion mutants (Pcr- PhoA-) and TnphoA fusion mutants (PhoA+) were isolated and screened for the ability to invade cultured HEp-2 cells. Most invasion-negative (Inv-) mutations mapped to a 30-kb segment of the invasion plasmid, including homologs of the Shigella flexneri ipa, mxi, and spa genes. Inv- PhoA+ fusions in the EIEC ipaC, mxiG, mxiJ, mxiM, and mxiD homologs and in a proposed new gene, named invX, located downstream of the spa region were identified and characterized. This analysis indicates the presence of the ipaC, mxiG, mxiJ, mxiM, mxiD, and invX gene products in the EIEC cell envelope and demonstrates a strict requirement for these genetic loci in invasion. Overall, our results suggest a high degree of genetic, structural, and functional homology between the EIEC and S. flexneri large invasion plasmids.

The immunological and cellular properties of cultured normal and fetal brain cells as well as glioma cells were compared. They were grown successfully in tissue culture media. Results from the growth properties and karyotype analysis indicated that cultured cells from normal and fetal brain tissues were normal and could be passaged limited times. The fetal brain cells had a longer life span than normal brain cells in the culture and their morphology exhibited variations according to cell passages. Two glioma cell lines, designated as G-5-T and G-9-T were established. The G-5-T and G-9-T had different morphology. Both G-5-T and G-9-T formed colonies in the soft agar. However, only G-9-T cells grew as large tumors in nude mice. Neither cell line secreted CEA, AFP and did not contain GFAP and S-100 protein. As measured by the 51Cr cytotoxicity assay, G-9-T but not G-5-T cells possessed D/DR antigens.