Leveraging vaccination-induced protective antibodies to define conserved epitopes on influenza N2 neuraminidase.

There is growing appreciation for neuraminidase (NA) as an influenza vaccine target; however, its antigenicity remains poorly characterized. In this study, we isolated three broadly reactive N2 antibodies from the plasmablasts of a single vaccinee, including one that cross-reacts with NAs from seasonal H3N2 strains spanning five decades. Although these three antibodies have diverse germline usages, they recognize similar epitopes that are distant from the NA active site and instead involve the highly conserved underside of NA head domain. We also showed that all three antibodies confer prophylactic and therapeutic protection in vivo, due to both Fc effector functions and NA inhibition through steric hindrance. Additionally, the contribution of Fc effector functions to protection in vivo inversely correlates with viral growth inhibition activity in vitro. Overall, our findings advance the understanding of NA antibody response and provide important insights into the development of a broadly protective influenza vaccine.

Chimeric hemagglutinin split vaccines elicit broadly cross-reactive antibodies and protection against group 2 influenza viruses in mice.

Seasonal influenza virus vaccines are effective when they are well matched to circulating strains. Because of antigenic drift/change in the immunodominant hemagglutinin (HA) head domain, annual vaccine reformulations are necessary to maintain a match with circulating strains. In addition, seasonal vaccines provide little to no protection against newly emerging pandemic strains. Sequential vaccination with chimeric HA (cHA) constructs has been proven to direct the immune response toward the immunosubdominant but more conserved HA stalk domain. In this study, we show that immunization with group 2 cHA split vaccines in combination with the CpG 1018 adjuvant elicits broadly cross-reactive antibodies against all group 2 HAs, as well as systemic and local antigen-specific T cell responses. Antibodies elicited after sequential vaccination are directed to conserved regions of the HA such as the stalk and the trimer interface and also to the N2 neuraminidase (NA). Immunized mice were fully protected from challenge with a broad panel of influenza A viruses.

Activation of Toll-like receptor 4 by Ebola virus-shed glycoprotein is direct and requires the internal fusion loop but not glycosylation.

Infection by the Ebola virus, a member of the Filoviridae family of RNA viruses, leads to acute viral hemorrhagic fever. End-stage Ebola virus disease is characterized by a cytokine storm that causes tissue damage, vascular disintegration, and multi-organ failure. Previous studies showed that a shed form of the viral spike glycoprotein (sGP1,2) drives this hyperinflammatory response by activating Toll-like receptor 4 (TLR4). Here, we find that glycosylation is not required for activation of TLR4 by sGP1,2 and identify the internal fusion loop (IFL) as essential for inflammatory signaling. sGP1,2 competes with lipid antagonists of TLR4, and the IFL interacts directly with TLR4 and co-receptor MD2. Together, these findings indicate that sGP1,2 activates TLR4 analogously to bacterial agonist lipopolysaccharide (LPS) by binding into a hydrophobic pocket in MD2 and promoting the formation of an active heterotetramer. This conclusion is supported by docking studies that predict binding sites for sGP1,2 on TLR4 and MD2.

Freshwater monitoring by nanopore sequencing.

While traditional microbiological freshwater tests focus on the detection of specific bacterial indicator species, including pathogens, direct tracing of all aquatic DNA through metagenomics poses a profound alternative. Yet, in situ metagenomic water surveys face substantial challenges in cost and logistics. Here, we present a simple, fast, cost-effective and remotely accessible freshwater diagnostics workflow centred around the portable nanopore sequencing technology. Using defined compositions and spatiotemporal microbiota from surface water of an example river in Cambridge (UK), we provide optimised experimental and bioinformatics guidelines, including a benchmark with twelve taxonomic classification tools for nanopore sequences. We find that nanopore metagenomics can depict the hydrological core microbiome and fine temporal gradients in line with complementary physicochemical measurements. In a public health context, these data feature relevant sewage signals and pathogen maps at species level resolution. We anticipate that this framework will gather momentum for new environmental monitoring initiatives using portable devices.

Many water-dwelling bacteria can cause severe diseases such as cholera, typhoid or leptospirosis. One way to prevent outbreaks is to test water sources to find out which species of microbes they contain, and at which levels. Traditionally, this involves taking a water sample, followed by growing a few species of 'indicator bacteria' that help to estimate whether the water is safe. An alternative technique, called metagenomics, has been available since the mid-2000s. It consists in reviewing (or 'sequencing') the genetic information of most of the bacteria present in the water, which allows scientists to spot harmful species. Both methods, however, require well-equipped laboratories with highly trained staff, making them challenging to use in remote areas. The MinION is a pocket-sized device that ­ when paired with a laptop or mobile phone ­ can sequence genetic information 'on the go'. It has already been harnessed during Ebola, Zika or SARS-CoV-2 epidemics to track the genetic information of viruses in patients and environmental samples. However, it is still difficult to use the MinION and other sequencers to monitor bacteria in water sources, partly because the genetic information of the microbes is highly fragmented during DNA extraction. To address this challenge, Urban, Holzer et al. set out to optimise hardware and software protocols so the MinION could be used to detect bacterial species present in rivers. The tests focussed on the River Cam in Cambridge, UK, a waterway which faces regular public health problems: local rowers and swimmers often contract waterborne infections, sometimes leading to river closures. For six months, Urban, Holzer et al. used the MinION to map out the bacteria present across nine river sites, assessing the diversity of species and the presence of disease-causing microbes in the water. In particular, the results showed that optimising the protocols made it possible to tell the difference between closely related species ­ an important feature since harmful and inoffensive bacteria can sometimes be genetically close. The data also revealed that the levels of harmful bacteria were highest downstream of urban river sections, near a water treatment plant and river barge moorings. Together, these findings demonstrate that optimising MinION protocols can turn this device into a useful tool to easily monitor water quality. Around the world, climate change, rising urbanisation and the intensification of agriculture all threaten water quality. In fact, access to clean water is one of the United Nations sustainable development goals for 2030. Using the guidelines developed by Urban, Holzer et al., communities could harness the MinION to monitor water quality in remote areas, offering a cost-effective, portable DNA analysis tool to protect populations against deadly diseases.

Negative Regulation of TLR Signaling by BCAP Requires Dimerization of Its DBB Domain.

The B cell adaptor protein (BCAP) is a multimodular regulator of inflammatory signaling in diverse immune system cells. BCAP couples TLR signaling to phosphoinositide metabolism and inhibits MyD88-directed signal transduction. BCAP is recruited to the TLR signalosome forming multitypic interactions with the MAL and MyD88 signaling adaptors. In this study, we show that indirect dimerization of BCAP TIR is required for negative regulation of TLR signaling. This regulation is mediated by a transcription factor Ig (TIG/IPT) domain, a fold found in the NF-κB family of transcription factors. We have solved the crystal structure of the BCAP TIG and find that it is most similar to that of early B cell factor 1 (EBF1). In both cases, the dimer is stabilized by a helix-loop-helix motif at the C terminus and interactions between the ß-sheets of the Ig domains. BCAP is exclusively localized in the cytosol and is unable to bind DNA. Thus, the TIG domain is a promiscuous dimerization module that has been appropriated for a range of regulatory functions in gene expression and signal transduction.

Mutual reporting of process and outcomes enhances quality outcomes for colon and rectal resections.

BACKGROUND: This report describes the favorable results of mutual reporting of process and outcome of care for major resections of the colon and rectum, one of six focal points for the Center for Medicare Services studies seeking to broadly reduce death and complications and enhance consistency of care. METHODS: A group of 66 surgical specialists in 9 cities in Kentucky reported cases to a quality improvement network over the past 5 years, and these data were supplemented by chart verification and patient satisfaction surveys. Consecutive colon and rectal resections (N=309) were reported by 23 general and colorectal surgeons. Eighty percent of the operations were performed by 4 surgeons. RESULTS: Forty-four percent of the patients had colorectal cancer, and 27% had diverticulitis; 84% of colon resections were performed by general surgeons whereas 77% of rectal resections were performed by colorectal specialists. Audit showed 6 leaks/fistulas and 16 patients who required unscheduled readmissions. Eleven patients had prolonged ileus. Only 2 patients died. Consensus among network surgeons included the following: 1. Mutual reporting led to a narrowing of choices and improved timing for antibiotic prophylaxis. 2. Standard order sets in one hospital led to a shortened duration of stay. 3. Surgeon participation in a quality improvement network led to a safe reduction in preoperative cardiology consultation. 4. More patients arrive with all evaluations complete due to increased utilization of preoperative anesthesiology clinics. 5. Enhanced operating room throughput has been achieved by joint anesthesia/surgery reporting and includes reduced time to induction of anesthesia and in the Post-Anesthesia Care Unit and lessened use of expensive postoperative antiemetics. 6. Reported medication errors were reduced by standard order sets, as were other reported adverse events. CONCLUSIONS: Practicing surgeons meet and/or exceed published benchmarks for colorectal resections and can further improve their outcomes by standardization and refinement of orders and procedures and improved collaboration with anesthesiologists.

Splenic Artery Avulsion in a High School Football Player: A Case Report.

OBJECTIVE: To present the case of a high school football player who sustained avulsion of 2 branches of the splenic artery from his spleen as he was tackled and landed on the football. BACKGROUND: A high school football player was tackled and fell onto the football, left side first. He was examined by a certified athletic trainer and an internist. On evaluation, he had a positive Kehr sign, exquisite left upper abdominal quadrant tenderness, and complaint of nausea. He also exhibited signs of the onset of shock, including diaphoresis, a rapid pulse, and hypotension. He was immediately transported by ambulance to the local emergency facility. DIFFERENTIAL DIAGNOSIS: Splenic rupture, splenic laceration, splenic artery avulsion, or ruptured viscus. TREATMENT: Emergency surgery was performed, with removal of 2800 mL of blood and ligation of the 2 arterial branches avulsed from the spleen. The patient fully recovered within 6 weeks and was cleared to resume all sports activities. UNIQUENESS: Injury to the spleen in football is a known yet very uncommon injury. Even more unusual is the avulsion of splenic artery branches from the spleen. CONCLUSIONS: It is critical that athletic trainers and team physicians have an understanding of the mechanisms, signs, and symptoms of splenic injury. Because the spleen is a highly vascular organ, severe hemorrhage can be fatal in just minutes if not recognized and appropriately treated.