Superior immunogenicity of HCV envelope glycoproteins when adjuvanted with cyclic-di-AMP, a STING activator or archaeosomes.

Three decades after the discovery, hepatitis C virus (HCV) is still the leading cause of liver transplantation and poses a major threat to global health. In spite of recent advances in the development of direct acting antivirals, there is still a need for a prophylactic vaccine to limit the virus spread and protect at-risk populations, especially in developing countries, where the cost of the new treatments may severely limit access. The use of recombinant HCV glycoproteins E1E2 (rE1E2) in combination with the MF59, an oil-in-water emulsion-based adjuvant, has previously been shown to reduce the rate of chronicity in chimpanzees and to induce production of cross-neutralizing antibodies and cellular immune responses in human volunteers. To further improve neutralizing antibody responses in recipients along with robust T cell responses, we have explored the immunogenicity of different adjuvants when formulated with the HCV rE1E2 vaccine in mice. Our data show that cyclic di-adenosine monophosphate (c-di-AMP) and archaeosomes elicit strong neutralizing antibodies similar to those elicited using aluminum hydroxide/monophosphoryl lipid A (Alum/monophos. /MPLA) and MF59. However, both c-di-AMP and archaeosomes induced a more robust cellular immune response, which was confirmed by the detection of vaccine-specific poly-functional CD4+ T cells. We conclude that these adjuvants may substantially boost the immunogenicity of our E1E2 vaccine. In addition, our data also indicates that use of a partial or exclusive intranasal immunization regimen may also be feasible using c-di-AMP as adjuvant.

Standardized methods to generate mock (spiked) clinical specimens by spiking blood or plasma with cultured pathogens.

AIMS: To demonstrate standardized methods for spiking pathogens into human matrices for evaluation and comparison among diagnostic platforms. METHODS AND RESULTS: This study presents detailed methods for spiking bacteria or protozoan parasites into whole blood and virus into plasma. Proper methods must start with a documented, reproducible pathogen source followed by steps that include standardized culture, preparation of cryopreserved aliquots, quantification of the aliquots by molecular methods, production of sufficient numbers of individual specimens and testing of the platform with multiple mock specimens. Results are presented following the described procedures that showed acceptable reproducibility comparing in-house real-time PCR assays to a commercially available multiplex molecular assay. CONCLUSIONS: A step by step procedure has been described that can be followed by assay developers who are targeting low prevalence pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of diagnostic platforms for detection of low prevalence pathogens such as biothreat or emerging agents is challenged by the lack of clinical specimens for performance evaluation. This deficit can be overcome using mock clinical specimens made by spiking cultured pathogens into human matrices. To facilitate evaluation and comparison among platforms, standardized methods must be followed in the preparation and application of spiked specimens.

Septic arthritis of the hip secondary to rat bite fever: a case report.

Rat bite fever is a rare infection typically caused by Streptobacillus moniliformis. The mode of transmission is most commonly through a bite or scratch from an infected rat. This disease is characterized by polyarthritis, fever, and a delayed onset erythematous maculopapular rash of the extremities. The authors report a case of rat bite fever, which led to septic arthritis of the hip. To the authors' knowledge, the complication of hip sepsis requiring an arthrotomy has not been reported in the literature. The orthopaedist should be aware of not only Streptobacillus moniliformis, but also of other zoonotic organisms, which potentially can cause septic arthritis and warrant treatment with specific antibiotics.

A review of progress towards elucidating the role of protein kinase CK2 in polymerase III transcription: regulation of the TATA binding protein.

We have investigated the molecular basis of the requirement for protein kinase CK2 in nuclear transcription in Saccharomyces cerevisiae. In vivo and in vitro analysis has demonstrated that CK2 is required for efficient transcription of the tRNA and 55 rRNA genes by RNA polymerase III. This suggests that a component of the pol III transcription machinery is regulated by CK2. We tested this possibility by a biochemical complementation approach in which components of the pol III transcription machinery from wild type cells were tested for their ability to rescue transcription in extract from a conditionally CK2-deficient mutant. We found that pol III transcription initiation factor IIIB (TFIIIB) fully restores transcription in CK2-deficient extract. Further in vitro studies revealed that TFIIIB must be phosphorylated to be active, that a single subunit of wild type TFIIIB, the TATA binding protein (TBP), is efficiently phosphorylated by CK2, and that recombinant TBP and a limiting amount of CK2 rescues transcription in CK2-deficient extract. We conclude that TBP is the physiological target of CK2 among the components of the pol III transcription machinery. The implications of this result are discussed in the context of previous data concerning the regulation of TFIIIB.

Benzalkonium chloride. A potential disinfecting irrigation solution for orthopaedic wounds.

The efficacy of benzalkonium chloride was evaluated as an irrigating solution for the eradication of Staphylococcus aureus from a contaminated orthopaedic wound. Thirty Sprague Dawley rats were randomized into two groups. A stainless steel wire was placed in a lumbar spinous process, and the wound was inoculated with 10(7) or 10(6) colony forming units of Staphylococcus aureus. The wound was irrigated with 1 L of normal saline or 0.1% benzalkonium chloride solution. The animals were sacrificed, and cultures were obtained. Rats inoculated with 10(7) colony forming units of Staphylococcus aureus and irrigated with benzalkonium chloride had a significant decrease in the total number of positive cultures, deep wound cultures, and stainless steel wire cultures. Rats inoculated with 10(6) colony forming units of Staphylococcus aureus and irrigated with benzalkonium chloride also had a significant decrease in the total number of positive cultures, deep wound cultures, and stainless steel wire cultures. In a parallel noninoculation study, histologic evaluation of tissues did not show toxicity in the rats irrigated with benzalkonium chloride. This study shows that benzalkonium chloride is more effective than normal saline as an irrigating agent for eradicating Staphylococcus aureus from a contaminated orthopaedic wound.

Chromatin assembly in a yeast whole-cell extract.

A simple in vitro system that supports chromatin assembly was developed for Saccharomyces cerevisiae. The assembly reaction is ATP-dependent, uses soluble histones and assembly factors, and generates physiologically spaced nucleosomes. We analyze the pathway of histone recruitment into nucleosomes, using this system in combination with genetic methods for the manipulation of yeast. This analysis supports the model of sequential recruitment of H3/H4 tetramers and H2A/H2B dimers into nucleosomes. Using a similar approach, we show that DNA ligase I can play an important role in template repair during assembly. These studies demonstrate the utility of this system for the combined biochemical and genetic analysis of chromatin assembly in yeast.

Benzalkonium chloride: a potential disinfecting irrigation solution.

OBJECTIVE: To determine the disinfecting properties of benzalkonium chloride as an irrigation agent. DESIGN: Comparison was made between irrigation of contaminated muscle strips with benzalkonium chloride and normal saline (control). SUMMARY OF BACKGROUND DATA: Benzalkonium chloride is a cationic disinfectant, which has questionable efficacy in an organic environment. However, no previous study has attempted to use high volumes of this cationic solution to overcome the neutralizing effect of organic tissue and thus maintain this detergent's germicidal properties. METHODS: 2.5 cm x 0.5 cm x 0.5 cm pieces of bovine muscle were aseptically cut from the center of freshly harvested beef muscle and incubated with 1.0 x 10(7) colony forming units of bacteria for 15 minutes. The muscle strips were then irrigated with either 100 mL, 1 L, or 10 L of benzalkonium chloride at a 1:2000 concentration in normal saline. Normal saline was used as the control. The muscle strips were sonicated to remove adherent bacteria; the number of living organisms was determined by quantitatively culturing the sonicate. RESULTS: In vitro, benzalkonium chloride was superior to normal saline at disinfecting bovine muscle (p < or = 0.001). When 10 L of benzalkonium chloride irrigation was used, no living bacteria could be recovered (p < or = 0.012). CONCLUSION: In this experimental setting benzalkonium chloride was an effective disinfection agent, with enhanced activity at large volumes.

Casein kinase II is required for efficient transcription by RNA polymerase III.

Casein kinase II (CKII) is a ubiquitous and highly conserved serine/threonine protein kinase found in the nucleus and cytoplasm of most cells. Using a combined biochemical and genetic approach in the yeast Saccharomyces cerevisiae, we assessed the role of CKII in specific transcription by RNA polymerases I, II, and III. CKII is not required for basal transcription by RNA polymerases I and II but is important for polymerase III transcription. Polymerase III transcription is high in extracts with normal CKII activity but low in extracts from a temperature-sensitive mutant that has decreased CKII activity due to a lesion in the enzyme's catalytic alpha' subunit. Polymerase III transcription of 5S rRNA and tRNA templates in the temperature-sensitive extract is rescued by purified, wild-type CKII. An inhibitor of CKII represses polymerase III transcription in wild-type extract, and this repression is partly overcome by supplementing reaction mixtures with active CKII. Finally, we show that polymerase III transcription in vivo is impaired when CKII is inactivated. Our results demonstrate that CKII, an oncogenic protein kinase previously implicated in cell cycle and growth control, is required for high-level transcription by RNA polymerase III.

Intragenic suppression of integration-defective IS10 transposase mutants.

IS10 transposase mediates excision and integration reactions in Tn10/IS10 transposition. Mutations in IS10 transposase that specifically block integration have previously been identified; however, the mechanism by which these mutations block integration has not been established. One approach to defining the basis of this block is to identify ways in which the original defect can be corrected. The approach we have taken toward this end has been to isolate and characterize intragenic second site suppressors to two different integration-defective mutants. Of the second site suppressors identified, one, CY134, is of particular interest for two reasons. First, it suppresses at least seven different mutations that confer an integration-defective phenotype. Interestingly, these mutations map in two separate segments of transposase, designated patch I and patch II. Second, CY134 on its own has previously been shown to relax the target DNA sequence requirements for Tn10 integration. We provide evidence that suppression by CY134 is not simply a consequence of this mutation conferring a general "transposition up" phenotype, but rather is due to correcting the original defect. Possible mechanisms of suppression for both CY134 and other second site suppressors are considered.