An Agitated Patient With COVID-19 Infection and Early-onset Alzheimer Disease.

Encephalopathy, delirium, and agitation are documented symptoms of coronavirus disease (COVID-19) infection, but research into the management of agitation in the setting of COVID-19 and pre-existing neuropsychiatric disease is ongoing. We present a 55-year-old male patient with early-onset Alzheimer disease and deteriorating mental and functional status who presented to our institution with agitation and persistent COVID-19 positivity on polymerase chain reaction testing. His agitation was improved through pharmacologic optimization including the avoidance of benzodiazepines and initiation of clonidine and prazosin, which temporally coincided with the resolution of his nearly 2-month long COVID-19 positivity.

Successful removal of a 16 mm long pulp stone using ultrasonic tips from maxillary left first molar and its endodontic management.

To present a case report describing successful removal of a 16 mm long pulp stone in a single mass from a maxillary left first molar and its endodontic management. In the presented case, the conventional approach of dissecting the pulp stone was not considered, as radiographically the calcification was extending up to the apex of the palatal root canal and the separation of the coronal and radicular pulp stone would have risked the obliteration of its orifice. A new set of ultrasonic endodontic tips were used for the removal of significantly large pulp stone in single mass.

Peptide nucleic acids inhibit growth of Brucella suis in pure culture and in infected murine macrophages.

Peptide nucleic acids (PNAs) are single-stranded, synthetic nucleic acid analogues containing a pseudopeptide backbone in place of the phosphodiester sugar-phosphate. When PNAs are covalently linked to cell-penetrating peptides (CPPs) they readily penetrate the bacterial cell envelope, inhibit expression of targeted genes and cause growth inhibition both of Gram-positive and Gram-negative bacteria. However, the effectiveness of PNAs against Brucella, a facultative intracellular bacterial pathogen, was unknown. The susceptibility of a virulent Brucella suis strain to a variety of PNAs was assessed in pure culture as well as in murine macrophages. The studies showed that some of the PNAs targeted to Brucella genes involved in DNA (polA, dnaG, gyrA), RNA (rpoB), cell envelope (asd), fatty acid (kdtA, acpP) and protein (tsf) synthesis inhibit the growth of B. suis in culture and in macrophages after 24 h of treatment. PNA treatment inhibited Brucella growth by interfering with gene expression in a sequence-specific and dose-dependent manner at micromolar concentrations. The most effective PNA in broth culture was that targeting polA at ca. 12 µM. In contrast, in B. suis-infected macrophages, the most effective PNAs were those targeting asd and dnaG at 30 µM; both of these PNAs had little inhibitory effect on Brucella in broth culture. The polA PNA that inhibits wild-type B. suis also inhibits the growth of wild-type Brucella melitensis 16M and Brucella abortus 2308 in culture. This study reveals the potential usefulness of antisense PNA constructs as novel therapeutic agents against intracellular Brucella.

Magnetic Nanoclusters with Hydrophilic Spacing for Dual Drug Delivery and Sensitive Magnetic Resonance Imaging.

Magnetic Block Ionomer Clusters (MBIClusters) with hydrophilic ionic cores and nonionic coronas have been prepared that have ultrahigh transverse NMR relaxivities together with capacities for incorporating high concentrations of polar antibiotic payloads. Magnetite-polymer nanoparticles were assembled by adsorbing the polyacrylate block of an aminofunctional poly(ethylene oxide-b-acrylate) (H2N-PEO-b-PAA) copolymer onto magnetite nanoparticles. The PEO blocks extended into aqueous media to keep the nanoparticles dispersed. Amines at the tips of the H2N-PEO corona were then linked through reaction with a PEO diacrylate oligomer to yield MBIClusters where the metal oxide in the precursor nanoparticles were distinctly separated by the hydrophilic polymer. The intensity average spacing between the magnetite nanoparticles within the clusters was estimated to be ~50 nm. These MBIClusters with hydrophilic intra-cluster space had transverse relaxivities (r2's) that increased from 190 to 604 s-1 mM Fe-1 measured at 1.4 T and 37 °C as their average sizes increased. The clusters were loaded with up to ~38 wt% of the multi-cationic drug gentamicin. MRI scans focused on the livers of mice demonstrated that these MBIClusters are sensitive contrast agents.

Effect of exogenous erythritol on growth and survival of Brucella.

Erythritol has been considered as an important factor for the pathogenesis of Brucella abortus 2308 and its ability to cause abortion in ruminants. There is a lack of laboratory models to study the Brucella-erythritol relationship, as commonly used murine models do not have erythritol. We tested the effect of exogenous erythritol on the growth of Brucella in iron minimal medium (IMM), in infected macrophage culture and in infected mice to determine if these models can be used to study the relationship between Brucella and erythritol. An effect of erythritol on Brucella growth was only seen in IMM. There appear to be no effect of erythritol on Brucella growth in macrophage cell cultures or in mice. This shows that administration of erythritol to the mice or macrophages cannot mimic the environment in ruminants during pregnancy and thus cannot be used as models to understand the effect of erythritol on Brucella pathogenesis.

Characterization of outer membrane vesicles from Brucella melitensis and protection induced in mice.

The outer membrane vesicles (OMVs) from smooth B. melitensis 16 M and a derived rough mutant, VTRM1 strain, were purified and characterized with respect to protein content and induction of immune responses in mice. Proteomic analysis showed 29 proteins present in OMVs from B. melitensis 16 M; some of them are well-known Brucella immunogens such as SOD, GroES, Omp31, Omp25, Omp19, bp26, and Omp16. OMVs from a rough VTRM1 induced significantly higher expression of IL-12, TNFα, and IFNγ genes in bone marrow dendritic cells than OMVs from smooth strain 16 M. Relative to saline control group, mice immunized intramuscularly with rough and smooth OMVs were protected from challenge with virulent strain B. melitensis 16 M just as well as the group immunized with live strain B. melitensis Rev1 (P < 0.005). Additionally, the levels of serum IgG2a increased in mice vaccinated with OMVs from rough strain VTRM1 consistent with the induction of cell-mediated immunity.

Effect of entF deletion on iron acquisition and erythritol metabolism by Brucella abortus 2308.

Brucella abortus has been shown to produce two siderophores: 2,3-dihydroxybenzoic acid (2,3-DHBA) and brucebactin. Previous studies on Brucella have shown that 2,3-DHBA is associated with erythritol utilization and virulence in pregnant ruminants. The biosynthetic pathway and role of brucebactin are not known and the only gene shown to be involved so far is entF. Using cre-lox methodology, an entF mutant was created in wild-type B. abortus 2308. Compared with the wild-type strain, the ΔentF strain showed significant growth inhibition in iron minimal media that became exacerbated in the presence of an iron chelator. For the first time, we have demonstrated the death of the ΔentF strain under iron-limiting conditions in the presence of erythritol. Addition of FeCl(3) restored the growth of the ΔentF strain, suggesting a significant role in iron acquisition. Further, complementation of the ΔentF strain using a plasmid containing an entF gene suggested the absence of any polar effects. In contrast, there was no significant difference in survival and growth between the ΔentF and wild-type strains grown in the murine macrophage cell line J774A.1, suggesting that an alternate iron acquisition pathway is present in Brucella when grown intracellulary.

Immunization of mice with gamma-irradiated Brucella neotomae and its recombinant strains induces protection against virulent B. abortus, B. melitensis, and B. suis challenge.

Human brucellosis, a zoonotic disease of major public health concern in several developing countries, is primarily caused by Brucella abortus, Brucella melitensis, and Brucella suis. No brucellosis vaccine is available for human use. The aim of this study was to determine if Brucella neotomae, a bacterium not known to cause disease in any host, can be used for developing brucellosis vaccines. B. neotomae and its recombinant strains overexpressing superoxide dismutase and a 26 kDa periplasmic protein were rendered non-replicative through exposure to gamma-radiation and used as vaccines in a murine brucellosis model. All three vaccines induced antigen-specific antibody and T cell responses. The vaccinated mice showed significant resistance against challenge with virulent B. abortus 2308, B. melitensis 16 M, and B. suis 1330. These results demonstrate that the avirulent B. neotomae is a promising platform for developing a safe and effective vaccine for human brucellosis.

Identification of a single-nucleotide insertion in the promoter region affecting the sodC promoter activity in Brucella neotomae.

Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD), a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli ß-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.

Targeting Brucella melitensis with polymeric nanoparticles containing streptomycin and doxycycline.

Treatment and eradication of intracellular pathogens such as Brucella is difficult because infections are localized within phagocytic cells and most antibiotics, although highly active in vitro, do not actively pass through cellular membranes. Thus, an optimum strategy to treat these infections should address targeting of active drugs to the intracellular compartment where the bacteria replicate, and should prolong the release of the antibiotics so that the number of doses and associated toxicity can be reduced. We incorporated streptomycin and doxycycline into macromolecular nanoplexes with anionic homo- and block copolymers via cooperative electrostatic interactions among the cationic drugs and anionic polymers. The approach enabled simultaneous binding of both antibiotics into the nanoplexes, and their use resulted in an improvement in performance as compared with the free drugs. Administration of two doses of the nanoplexes significantly reduced the Brucella melitensis load in the spleens and livers of infected BALB/c mice. The nanoplexes were more effective than free drugs in the spleens (0.72-log and 0.51-log reductions, respectively) and in the livers (0.79-log and 0.42-log reductions, respectively) of the infected mice. Further research regarding the design of optimum nanoplex structures will be directed towards alterations in both the core and the shell properties to investigate the effects of the rates and pathways of entry into immune cells where the brucellae replicate.

Activity of native vs. synthetic promoters in Brucella.

Brucellosis caused by Brucella species is reportedly the most common zoonotic infection worldwide. The bacterial pathogen is also classified by the Centers for Disease Control and Prevention as a category (B) pathogen that has the potential for development as a bioweapon. Although eight genomes of Brucella have been sequenced, little information is available regarding the regulation of gene expression and promoter activity in Brucella spp. We therefore constructed a set of broad-host-range vectors expressing the lacZ reporter gene from various promoters. Four groups of promoters (Brucella native, antibiotic resistant, bacteriophage and synthetic promoters) were tested in vivo and in vitro in Brucella suis. The highest level of heterologous gene expression was achieved with synthetic hybrid trc promoter carrying the adenine-rich upstream element. Furthermore, this demonstrates the usefulness of synthetic promoters for enhanced level of gene expression in Brucella spp.

Prevention of vertical transmission of Neospora caninum in C57BL/6 mice vaccinated with Brucella abortus strain RB51 expressing N. caninum protective antigens.

Bovine abortions caused by the apicomplexan parasite Neospora caninum have been responsible for severe economic losses to the cattle industry. Infected cows either experience abortion or transmit the parasite transplacentally at a rate of up to 95%. Neospora caninum vaccines that can prevent vertical transmission and ensure disruption in the life cycle of the parasite greatly aid in the management of neosporosis in the cattle industry. Brucella abortus strain RB51, a commercially available vaccine for bovine brucellosis, can also be used as a vector to express plasmid-encoded proteins from other pathogens. Neospora caninum protective antigens MIC1, MIC3, GRA2, GRA6 and SRS2 were expressed in strain RB51. Female C57BL/6 mice were vaccinated with a recombinant strain RB51 expressing N. caninum antigen or irradiated tachyzoites, boosted 4 weeks later and then bred. Antigen-specific IgG, IFN-gamma and IL-10 were detected in vaccinated pregnant mice. Vaccinated mice were challenged with 5 x 10(6)N. caninum tachyzoites between days 11-13 of pregnancy. Brain tissue was collected from pups 3 weeks after birth and examined for the presence of N. caninum by real-time PCR. The RB51-MIC3, RB51-GRA6, irradiated tachyzoite vaccine, pooled strain RB51-Neospora vaccine, RB51-MIC1 and RB51-SRS2 vaccines elicited approximately 6-38% protection against vertical transmission. However, the differences in parasite burden in brain tissue of pups from the control and vaccinated groups were highly significant for all groups. Thus, B. abortus strain RB51 expressing the specific N. caninum antigens induced substantial protection against vertical transmission of N. caninum in mice.

Marek's disease virus encodes MicroRNAs that map to meq and the latency-associated transcript.

MicroRNAs (miRNAs) are a class of small (approximately 22-nucleotide) regulatory molecules that block translation or induce degradation of target mRNAs. These have been identified in a wide range of organisms, including viruses. In particular, the oncogenic gammaherpesviruses Kaposi's sarcoma herpesvirus and Epstein-Barr virus encode miRNAs that could potentially regulate either viral or host genes. To determine if Marek's disease virus (MDV), an oncogenic alphaherpesvirus of chickens, encodes miRNAs, we isolated small RNAs from MDV-infected chicken embryo fibroblasts (CEF) and used the 454 Life Sciences sequencing technology to obtain the sequences of 13,679 candidate host and viral small RNAs. Eight miRNAs were found, five of which flank the meq oncogene and three that map to the latency-associated transcript (LAT) region of the genome. The meq gene is unique to pathogenic serotypes of MDV and is transcriptionally active during latency and transformation, and the LAT region of the MDV genome is antisense to the immediate-early gene ICP4. Secondary structure analysis predicted that the regions flanking the miRNAs could form hairpin precursors. Northern blot analysis confirmed expression of all miRNAs in MDV-infected CEF, MDV-induced tumors, and MDV lymphoblastoid cell lines. We propose that the MDV miRNAs function to enable MDV pathogenesis and contribute to MDV-induced transformation of chicken T cells.

Symphyseal separation.

BACKGROUND: Separation of the pubic symphysis up to 1 cm during pregnancy and delivery occurs frequently. This report presents a woman who experienced a large symphyseal separation. CASE: Following delivery, a 35-year-old primipara complained of hip and groin pain associated with leg movement. An anterior-posterior pelvic X-ray showed a pubic separation of 9.5 cm and a 3-5 mm widening of the sacroiliac joints. She was treated with a pelvic binder, walker, and physical therapy. The diastasis has since undergone progressive reduction. CONCLUSION: Separation of the pubic symphysis during pregnancy and delivery is normal. However, a large separation is a potential complication requiring treatment and follow-up. Conservative management including analgesia, rest, and a pelvic binder is a reasonable method of management.