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The NlpC/p60-family of peptidoglycan hydrolases are key enzymes that facilitate bacterial cell division and also modulate microbe-host interactions. These endopeptidases utilize conserved Cys-His residues in their active site and are expressed in most bacterial species as well as some eukaryotes. Here we describe methods for biochemical analysis of Enterococcus faecium SagA-NlpC/p60 peptidoglycan hydrolase activity (Kim et al., 2019; Rangan et al., 2016), which includes recombinant protein preparation and biochemical analysis using both gel-based and LC-MS profiling of peptidoglycan fragments. These protocols should also facilitate the biochemical analysis of other NlpC/p60 peptidoglycan hydrolases.

An activated plasma contact system is an abnormality observed in many Alzheimer's disease (AD) patients. Since mild cognitive impairment (MCI) patients often develop AD, we analyzed the status of contact system activation in MCI patients. We found that kallikrein activity, high molecular weight kininogen cleavage, and bradykinin levels-measures of contact system activation-were significantly elevated in MCI patient plasma compared to plasma from ageand education matched healthy individuals. Changes were more pronounced in MCI patients with impaired short-term recall memory, indicating the possible role of the contact system in early cognitive changes.

Cancer immunotherapy has dramatically changed the approach to cancer treatment. The aim of targeting the immune system to recognize and destroy cancer cells has afforded many patients the prospect of achieving deep, long-term remission and potential cures. However, many challenges remain for achieving the goal of effective immunotherapy for all cancer patients. Checkpoint inhibitors have been able to achieve long-term responses in a minority of patients, yet improving response rates with combination therapies increases the possibility of toxicity. Chimeric antigen receptor T cells have demonstrated high response rates in hematological cancers, although most patients experience relapse. In addition, some cancers are notoriously immunologically "cold" and typically are not effective targets for immunotherapy. Overcoming these obstacles will require new strategies to improve upon the efficacy of current agents, identify biomarkers to select appropriate therapies, and discover new modalities to expand the accessibility of immunotherapy to additional tumor types and patient populations.

Leptopilina heterotoma are obligate parasitoid wasps that develop in the body of their Drosophila hosts. During oviposition, female wasps introduce venom into the larval hosts' body cavity. The venom contains discrete, 300 nm-wide, mixed-strategy extracellular vesicles (MSEVs), until recently referred to as virus-like particles. While the crucial immune suppressive functions of L. heterotoma MSEVs have remained undisputed, their biotic nature and origin still remain controversial. In recent proteomics analyses of L. heterotoma MSEVs, we identified 161 proteins in three classes: conserved eukaryotic proteins, infection and immunity related proteins, and proteins without clear annotation. Here we report 246 additional proteins from the L. heterotoma MSEV proteome. An enrichment analysis of the entire proteome supports vesicular nature of these structures. Sequences for more than 90% of these proteins are present in the whole-body transcriptome. Sequencing and de novo assembly of the 460 Mb-sized L. heterotoma genome revealed 90% of MSEV proteins have coding regions within the genomic scaffolds. Altogether, these results explain the stable association of MSEVs with their wasps, and like other wasp structures, their vertical inheritance. While our results do not rule out a viral origin of MSEVs, they suggest that a similar strategy for co-opting cellular machinery for immune suppression may be shared by other wasps to gain advantage over their hosts. These results are relevant to our understanding of the evolution of figitid and related wasp species.

Purpose of review Long-acting HIV treatment and prevention (LAHTP) can address some of the achievement gaps of daily oral therapy to bring us closer to achieving Joint United Nations Programme on HIV/AIDS Fast-track goals. Implementing these new technologies presents individual-level, population-level, and health systems-level opportunities and challenges. Recent findings To optimize LAHTP implementation and impact, decision-makers should define and gather relevant data to inform their investment case within the existing health systems context. Programmatic observations from scale-up of antiretroviral therapy, oral preexposure prophylaxis, voluntary medical male circumcision, and family planning offer lessons as planning begins for implementation of LAHTP. Additional data intelligence should be derived from formative studies, pragmatic clinical trials, epidemiologic and economic modeling of LAHTP. Key implementation issues that need to be addressed include optimal communication strategies for demand creation; target setting; logistics and supply chain of commodities needed for LAHTP delivery; human resource planning; defining and operationalizing monitoring and evaluating metrics; integration into health systems. Successful LAHTP implementation can bolster treatment and prevention coverage levels if implementation issues outlined above are proactively addressed in parallel with research and development so that health systems can more rapidly integrate new technologies as they gain regulatory approval.

Prokaryotes have developed numerous defense strategies to combat the constant threat posed by the diverse genetic parasites that endanger them. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas loci guard their hosts with an adaptive immune system against foreign nucleic acids. Protection starts with an immunization phase, in which short pieces of the invader's genome, known as spacers, are captured and integrated into the CRISPR locus after infection. Next, during the targeting phase, spacers are transcribed into CRISPR RNAs (crRNAs) that guide CRISPR-associated (Cas) nucleases to destroy the invader's DNA or RNA. Here we describe the many different molecular mechanisms of CRISPR targeting and how they are interconnected with the immunization phase through a third phase of the CRISPR-Cas immune response: primed spacer acquisition. In this phase, Cas proteins direct the crRNA-guided acquisition of additional spacers to achieve a more rapid and robust immunization of the population.

Background Validated, reliable, globally accepted outcome measurement instruments for hidradenitis suppurativa (HS) are needed. Current tools to measure the physical signs domain for HS rely on lesion counts, which are time-consuming and unreliable. Objectives To assess the reliability and validity of the Hidradenitis suppurativa Area and Severity Index Revised (HASI-R) tool, a novel method for assessing HS severity, incorporating signs of inflammation and body surface area involved. Methods The measurement properties of the HASI-R tool were evaluated. The tool was created by combining the previously published HASI and Severity and Area Score for Hidradenitis instruments. Twenty raters evaluated 15 patients with HS in a hospital-based ambulatory dermatology clinic. The objectives of the study were to assess inter- and intra-rater reliability of the HASI-R and its components, as well as its construct and known-groups validity. Existing lesion count-based clinician-reported measures of HS and their components were also assessed. Raters were also asked their preferences regarding the various HS severity assessment tools. Results The HASI-R had moderate inter-rater reliability [intra-class correlation coefficients (ICC) 0 center dot 60]. This was better than all other HS physical sign outcome measures evaluated, which had poor inter-rater reliability (ICC < 0 center dot 5). HASI-R had the highest intra-rater reliability (ICC 0 center dot 91). The HASI-R had good construct validity and demonstrated known-groups validity. The HASI-R was also the most preferred tool by all raters. Conclusions Results from the clinometric assessment of the HASI-R are encouraging, and support continued evaluation of this clinician-reported outcome measure.

The original version of this article unfortunately contained mistakes in reference numbers. The in-text citations and the references were mismatched. The original article has been corrected.