Omegasomes control formation, expansion, and closure of autophagosomes.

Autophagy, an essential cellular process for maintaining cellular homeostasis and eliminating harmful cytoplasmic objects, involves the de novo formation of double-membraned autophagosomes that engulf and degrade cellular debris, protein aggregates, damaged organelles, and pathogens. Central to this process is the phagophore, which forms from donor membranes rich in lipids synthesized at various cellular sites, including the endoplasmic reticulum (ER), which has emerged as a primary source. The ER-associated omegasomes, characterized by their distinctive omega-shaped structure and accumulation of phosphatidylinositol 3-phosphate (PI3P), play a pivotal role in autophagosome formation. Omegasomes are thought to serve as platforms for phagophore assembly by recruiting essential proteins such as DFCP1/ZFYVE1 and facilitating lipid transfer to expand the phagophore. Despite the critical importance of phagophore biogenesis, many aspects remain poorly understood, particularly the complete range of proteins involved in omegasome dynamics, and the detailed mechanisms of lipid transfer and membrane contact site formation.

Transient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells.

Genome editing by homology directed repair (HDR) is leveraged to precisely modify the genome of therapeutically relevant hematopoietic stem and progenitor cells (HSPCs). Here, we present a new approach to increasing the frequency of HDR in human HSPCs by the delivery of an inhibitor of 53BP1 (named "i53") as a recombinant peptide. We show that the use of i53 peptide effectively increases the frequency of HDR-mediated genome editing at a variety of therapeutically relevant loci in HSPCs as well as other primary human cell types. We show that incorporating the use of i53 recombinant protein allows high frequencies of HDR while lowering the amounts of AAV6 needed by 8-fold. HDR edited HSPCs were capable of long-term and bi-lineage hematopoietic reconstitution in NSG mice, suggesting that i53 recombinant protein might be safely integrated into the standard CRISPR/AAV6-mediated genome editing protocol to gain greater numbers of edited cells for transplantation of clinically meaningful cell populations.

Efficient and safe therapeutic use of paired Cas9-nickases for primary hyperoxaluria type 1.

The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. In addition, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.

CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.

The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.

ATPase-regulated autophagosome biogenesis.

Omega-shaped domains of the endoplasmic reticulum, known as omegasomes, have been suggested to contribute to autophagosome biogenesis, although their exact function is not known. Omegasomes are characterized by the presence of the double FYVE domain containing protein ZFYVE1/DFCP1, but it has remained a paradox that depletion of ZFYVE1 does not prevent bulk macroautophagy/autophagy. We recently showed that ZFYVE1 contains an N-terminal ATPase domain which dimerizes upon ATP binding. Mutations in the ATPase domain that inhibit ATP binding or hydrolysis do not prevent omegasome expansion and maturation. However, omegasome constriction is inhibited by these mutations, which results in an increased lifetime and thereby higher number of omegasomes. Interestingly, whereas ZFYVE1 knockout or mutations do not significantly affect bulk autophagy, selective autophagy of mitochondria, protein aggregates and micronuclei is inhibited. We propose that ATP binding and hydrolysis control the di- or multimerization state of ZFYVE1 which could provide the mechanochemical energy to drive large omegasome constriction and autophagosome completion.

Korea Seroprevalence Study of Monitoring of SARS-COV-2 Antibody Retention and Transmission (K-SEROSMART): findings from national representative sample.

OBJECTIVES: We estimated the population prevalence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including unreported infections, through a Korea Seroprevalence Study of Monitoring of SARS-CoV-2 Antibody Retention and Transmission (K-SEROSMART) in 258 communities throughout Korea. METHODS: In August 2022, a survey was conducted among 10,000 household members aged 5 years and older, in households selected through two stage probability random sampling. During face-to-face household interviews, participants self-reported their health status, COVID-19 diagnosis and vaccination history, and general characteristics. Subsequently, participants visited a community health center or medical clinic for blood sampling. Blood samples were analyzed for the presence of antibodies to spike proteins (anti-S) and antibodies to nucleocapsid proteins (anti-N) SARS-CoV-2 proteins using an electrochemiluminescence immunoassay. To estimate the population prevalence, the PROC SURVEYMEANS statistical procedure was employed, with weighting to reflect demographic data from July 2022. RESULTS: In total, 9,945 individuals from 5,041 households were surveyed across 258 communities, representing all basic local governments in Korea. The overall population-adjusted prevalence rates of anti-S and anti-N were 97.6% and 57.1%, respectively. Since the Korea Disease Control and Prevention Agency has reported a cumulative incidence of confirmed cases of 37.8% through July 31, 2022, the proportion of unreported infections among all COVID-19 infection was suggested to be 33.9%. CONCLUSIONS: The K-SEROSMART represents the first nationwide, community-based seroepidemiologic survey of COVID-19, confirming that most individuals possess antibodies to SARS-CoV-2 and that a significant number of unreported cases existed. Furthermore, this study lays the foundation for a surveillance system to continuously monitor transmission at the community level and the response to COVID-19.

A novel Cas9 fusion protein promotes targeted genome editing with reduced mutational burden in primary human cells.

Precise genome editing requires the resolution of nuclease-induced DNA double strand breaks (DSBs) via the homology-directed repair (HDR) pathway. In mammals, this is typically outcompeted by non-homologous end-joining (NHEJ) that can generate potentially genotoxic insertion/deletion mutations at DSB sites. Because of higher efficacy, clinical genome editing has been restricted to imperfect but efficient NHEJ-based approaches. Hence, strategies that promote DSB resolution via HDR are essential to facilitate clinical transition of HDR-based editing strategies and increase safety. Here we describe a novel platform that consists of a Cas9 fused to DNA repair factors to synergistically inhibit NHEJ and favor HDR for precise repairing of Cas-induced DSBs. Compared to canonical CRISPR/Cas9, the increase in error-free editing ranges from 1.5-fold to 7-fold in multiple cell lines and in primary human cells. This novel CRISPR/Cas9 platform accepts clinically relevant repair templates, such as oligodeoxynucleotides (ODNs) and adeno-associated virus (AAV)-based vectors, and has a lower propensity to induce chromosomal translocations as compared to benchmark CRISPR/Cas9. The observed reduced mutational burden, resulting from diminished indel formation at on- and off-target sites, provides a remarkable gain in safety and advocates this novel CRISPR system as an attractive tool for therapeutic applications depending on precision genome editing.

Genome Editing With TALEN, CRISPR-Cas9 and CRISPR-Cas12a in Combination With AAV6 Homology Donor Restores T Cell Function for XLP.

X-linked lymphoproliferative disease is a rare inherited immune disorder, caused by mutations or deletions in the SH2D1A gene that encodes an intracellular adapter protein SAP (Slam-associated protein). SAP is essential for mediating several key immune processes and the immune system - T cells in particular - are dysregulated in its absence. Patients present with a spectrum of clinical manifestations, including haemophagocytic lymphohistiocytosis (HLH), dysgammaglobulinemia, lymphoma and autoimmunity. Treatment options are limited, and patients rarely survive to adulthood without an allogeneic haematopoietic stem cell transplant (HSCT). However, this procedure can have poor outcomes in the mismatched donor setting or in the presence of active HLH, leaving an unmet clinical need. Autologous haematopoeitic stem cell or T cell therapy may offer alternative treatment options, removing the need to find a suitable donor for HSCT and any risk of alloreactivity. SAP has a tightly controlled expression profile that a conventional lentiviral gene delivery platform may not be able to fully replicate. A gene editing approach could preserve more of the endogenous regulatory elements that govern SAP expression, potentially providing a more optimum therapy. Here, we assessed the ability of TALEN, CRISPR-Cas9 and CRISPR-Cas12a nucleases to drive targeted insertion of SAP cDNA at the first exon of the SH2D1A locus using an adeno-associated virus serotype 6 (AAV6)-based vector containing the donor template. All nuclease platforms were capable of high efficiency gene editing, which was optimised using a serum-free AAV6 transduction protocol. We show that T cells from XLP patients corrected by gene editing tools have restored physiological levels of SAP gene expression and restore SAP-dependent immune functions, indicating a new therapeutic opportunity for XLP patients.

3D-Structured Illumination Microscopy of Centrosomes in Human Cell Lines.

The centrosome is the main microtubule-organizing center of animal cells, and is composed of two barrel-shaped microtubule-based centrioles embedded in protein dense pericentriolar material. Compositional and architectural re-organization of the centrosome drives its duplication, and enables its microtubule-organizing activity and capability to form the primary cilium, which extends from the mature (mother) centriole, as the cell exits the cell cycle. Centrosomes and primary cilia are essential to human health, signified by the causal role of centrosome- and cilia-aberrations in numerous congenic disorders, as well as in the etiology and progression of cancer. The list of disease-associated centrosomal proteins and their proximitomes is steadily expanding, emphasizing the need for high resolution mapping of such proteins to specific substructures of the organelle. Here, we provide a detailed 3D-structured illumination microscopy (3D-SIM) protocol for comparative localization analysis of fluorescently labeled proteins at the centrosome in fixed human cell lines, at approximately 120 nm lateral and 300 nm axial resolution. The procedure was optimized to work with primary antibodies previously known to depend on more disruptive fixation reagents, yet largely preserves centriole and centrosome architecture, as shown by transposing acquired images of landmark proteins on previously published transmission electron microscopy (TEM) images of centrosomes. Even more advantageously, it is compatible with fluorescent protein tags. Finally, we introduce an internal reference to ensure correct 3D channel alignment. This protocol hence enables flexible, swift, and information-rich localization and interdependence analyses of centrosomal proteins, as well as their disorder-associated mutations.

Technical aspects and operative nuances using a high-definition 4K-3-dimensional exoscope for carotid endarterectomy surgery.

PURPOSE: Carotid endarterectomy (CEA) is effective in treating carotid artery stenosis to prevent stroke. Historically, this operation has been performed utilizing loupe magnification with or without the operating microscope (OM). However, there remains a need for continued improvement in operative visualization and surgical ergonomics. Recently, newly developed digital 'exoscope' has provided the surgeon with unique lighting and magnification as well as improvements in surgical ergonomics and working angle. We sought to review our cumulative experience using a novel 4K high-definition (4K-HD) 3-dimensional (3D) exoscope (EX) for CEA surgery. METHODS: All CEA surgery cases at our institution between 2013 and 2019 using the 4K-HD 3D EX were reviewed. Operative parameters, patient outcome and operator's assessment of the EX compared to OM-assisted cases was conducted. RESULTS: 28 patients were treated, 10 of which were operated using the EX. All procedures were performed without perioperative complications, or significant differences in operative parameters (blood loss <20 cm3 and 164 ± 49.5 minutes) compared to OM-assisted cases. Operators reported improved level of comfort performing 'high' bifurcation surgery and improved visualization and posture during inspection of the distal ICA lumen as primary advantages of EX-assisted CEA over OM-assisted CEA. CONCLUSIONS: The ORBEYE EX, albeit a learning curve necessitating a short period of the OR team, provided safety and outcome comparable to OM-assisted surgery. Potential advantages noted were improved visualization and ergonomics specifically for when extreme working angles were required. Our experience suggests that the exoscope may become a valuable alternative to standard magnification tools in CEA surgery.

Targeted multi-epitope switching enables straightforward positive/negative selection of CAR T cells.

Chimeric antigen receptor (CAR) T cell technology has enabled successfully novel concepts to treat cancer patients, with substantial remission rates in lymphoid malignancies. This cell therapy is based on autologous T lymphocytes that are genetically modified to express a CAR that recognizes tumor-associated antigens and mediates the elimination of the respective tumor cells. Current limitations include laborious manufacturing procedures as well as severe immunological side effects upon administration of CAR T cells. To address these limitations, we integrated RQR8, a multi-epitope molecule harboring a CD34 epitope and two CD20 mimotopes, alongside a CD19-targeting CAR, into the CD52 locus. Using CRISPR-Cas9 and adeno-associated virus-based donor vectors, some 60% of genome-edited T cells were CAR+/CD20+/CD34+/CD52- without further selection. This could be increased to >95% purity after CD34 tag-based positive selection. These epitope-switched CAR T cells retained cell killing competence against CD19+ tumor cells, and were resistant to alemtuzumab (anti-CD52) but sensitive to rituximab (anti-CD20) in complement-dependent cytotoxicity assays. In conclusion, gene editing-based multiple epitope switching represents a promising development with the potential to improve both the manufacturing procedure as well as the clinical safety of CAR T cells.

Automated generation of gene-edited CAR T cells at clinical scale.

The potential of adoptive cell therapy can be extended when combined with genome editing. However, variation in the quality of the starting material and the different manufacturing steps are associated with production failure and product contamination. Here, we present an automated T cell engineering process to produce off-the-shelf chimeric antigen receptor (CAR) T cells on an extended CliniMACS Prodigy platform containing an in-line electroporation unit. This setup was used to combine lentiviral delivery of a CD19-targeting CAR with transfer of mRNA encoding a TRAC locus-targeting transcription activator-like effector nuclease (TALEN). In three runs at clinical scale, the T cell receptor (TCR) alpha chain encoding TRAC locus was disrupted in >35% of cells with high cell viability (>90%) and no detectable off-target activity. A final negative selection step allowed the generation of TCRα/ß-free CAR T cells with >99.5% purity. These CAR T cells proliferated well, maintained a T cell memory phenotype, eliminated CD19-positive tumor cells, and released the expected cytokines when exposed to B cell leukemia cells. In conclusion, we established an automated, good manufacturing practice (GMP)-compliant process that integrates lentiviral transduction with electroporation of TALEN mRNA to produce functional TCRα/ß-free CAR19 T cells at clinical scale.

ESCRT-mediated phagophore sealing during mitophagy.

Inactivation of the endosomal sorting complex required for transport (ESCRT) machinery has been reported to cause autophagic defects, but the exact functions of ESCRT proteins in macroautophagy/autophagy remain incompletely understood. Using live-cell fluorescence microscopy we found that the filament-forming ESCRT-III subunit CHMP4B was recruited transiently to nascent autophagosomes during starvation-induced autophagy and mitophagy, with residence times of about 1 and 2 min, respectively. Correlative light microscopy and electron tomography revealed CHMP4B recruitment at a late step in mitophagosome formation. The autophagosomal dwell time of CHMP4B was strongly increased by depletion of the regulatory ESCRT-III subunit CHMP2A. Using a novel optogenetic closure assay we observed that depletion of CHMP2A inhibited phagophore sealing during mitophagy. Consistent with this, depletion of CHMP2A and other ESCRT-III subunits inhibited both PRKN/PARKIN-dependent and -independent mitophagy. We conclude that the ESCRT machinery mediates phagophore closure, and that this is essential for mitophagic flux.Abbreviations: BSA: bovine serum albumin; CHMP: chromatin-modifying protein; CLEM: correlative light and electron microscopy; EGFP: enhanced green fluorescent protein; ESCRT: endosomal sorting complex required for transport; HEPES: 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; HRP: horseradish peroxidase; ILV: intralumenal vesicle; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; LOV2: light oxygen voltage 2; MLS: mitochondrial localization sequence; MT-CO2: mitochondrially encoded cytochrome c oxidase II; O+A: oligomycin and antimycin A; PBS: phosphate-buffered saline; PIPES: piperazine-N,N-bis(2-ethanesulfonic acid); PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RAB: RAS-related in brain; SD: standard deviation; SEM: standard error of the mean; TOMM20: TOMM20: translocase of outer mitochondrial membrane 20; VCL: vinculin; VPS4: vacuolar protein sorting protein 4; Zdk1: Zdark 1; TUBG: Tubulin gamma chain.

Tumor suppression by control of matrix metalloproteinase recycling.

Secretion of matrix metalloproteinases (MMPs) enables cancer cells to degrade extracellular matrix, thus promoting tumor invasion and metastasis. We have recently found that the endosomal protein WDFY2 serves as a gatekeeper for MMP recycling from endosomes and that deletion of WDFY2, which is frequently lost in metastatic cancers, causes increased matrix degradation and cell invasion.

A CEP104-CSPP1 Complex Is Required for Formation of Primary Cilia Competent in Hedgehog Signaling.

CEP104 is an evolutionarily conserved centrosomal and ciliary tip protein. CEP104 loss-of-function mutations are reported in patients with Joubert syndrome, but their function in the etiology of ciliopathies is poorly understood. Here, we show that cep104 silencing in zebrafish causes cilia-related manifestations: shortened cilia in Kupffer's vesicle, heart laterality, and cranial nerve development defects. We show that another Joubert syndrome-associated cilia tip protein, CSPP1, interacts with CEP104 at microtubules for the regulation of axoneme length. We demonstrate in human telomerase reverse transcriptase-immortalized retinal pigmented epithelium (hTERT-RPE1) cells that ciliary translocation of Smoothened in response to Hedgehog pathway stimulation is both CEP104 and CSPP1 dependent. However, CEP104 is not required for the ciliary recruitment of CSPP1, indicating that an intra-ciliary CEP104-CSPP1 complex controls axoneme length and Hedgehog signaling competence. Our in vivo and in vitro analyses of CEP104 define its interaction with CSPP1 as a requirement for the formation of Hedgehog signaling-competent cilia, defects that underlie Joubert syndrome.

Distinct roles of the anaphylatoxin receptors C3aR, C5aR1 and C5aR2 in experimental meningococcal infections.

The complement system is pivotal in the defense against invasive disease caused by Neisseria meningitidis (Nme, meningococcus), particularly via the membrane attack complex. Complement activation liberates the anaphylatoxins C3a and C5a, which activate three distinct G-protein coupled receptors, C3aR, C5aR1 and C5aR2 (anaphylatoxin receptors, ATRs). We recently discovered that C5aR1 exacerbates the course of the disease, revealing a downside of complement in Nme sepsis. Here, we compared the roles of all three ATRs during mouse nasal colonization, intraperitoneal infection and human whole blood infection with Nme. Deficiency of complement or ATRs did not alter nasal colonization, but significantly affected invasive disease: Compared to WT mice, the disease was aggravated in C3ar-/- mice, whereas C5ar1-/- and C5ar2-/- mice showed increased resistance to meningococcal sepsis. Surprisingly, deletion of either of the ATRs resulted in lower cytokine/chemokine responses, irrespective of the different susceptibilities of the mice. This was similar in ex vivo human whole blood infection using ATR inhibitors. Neutrophil responses to Nme were reduced in C5ar1-/- mouse blood. Upon stimulation with C5a plus Nme, mouse macrophages displayed reduced phosphorylation of ERK1/2, when C5aR1 or C5aR2 were ablated or inhibited, suggesting that both C5a-receptors prime an initial macrophage response to Nme. Finally, in vivo blockade of C5aR1 alone (PMX205) or along with C5aR2 (A8Δ71-73) resulted in ameliorated disease, whereas neither antagonizing C3aR (SB290157) nor its activation with a "super-agonist" peptide (WWGKKYRASKLGLAR) demonstrated a benefit. Thus, C5aR1 and C5aR2 augment disease pathology and are interesting targets for treatment, whereas C3aR is protective in experimental meningococcal sepsis.

Rechallenging Recurrent Glioblastoma with Intra-Arterial Bevacizumab with Blood Brain-Barrier Disruption Results in Radiographic Response.

BACKGROUND: High-dose bevacizumab delivered via super selective intra-arterial cerebral infusion (SIACI) is one promising clinical trial combination for patients with glioblastoma (GBM). Although both continuous intravenous and intra-arterial administration of bevacizumab, and rechallenge with intravenous bevacizumab, have demonstrated improved survival, this is the first description of rechallenging GBM with SIACI of bevacizumab. CASE DESCRIPTION: We report a case of a 43-year-old woman with recurrent GBM who had received treatment from 3 clinical trials, including a rechallenge with SIACI of bevacizumab. First, she enrolled into a phase I/II trial for patients newly diagnosed with GBM (NCT01811498) and received 3 doses of SIACI bevacizumab over 180 days in addition to standard of care chemotherapy and radiation. Following progression, as indicated on her magnetic resonance imaging scan, she consented for a separate clinical trial for her disease and received 2 cycles of temozolomide with an investigational agent. The patient was removed from the study on tumor progression. Subsequently, she was rechallenged with SIACI of bevacizumab via a third clinical trial (NCT01269853) and then completed 3 intravenous infusions. After completing the third trial, her magnetic resonance imaging scan demonstrated improvement based on Response Assessment In Neuro-Oncology criteria. CONCLUSIONS: This is the first report to highlight the effect of rechallenging a patient with SIACI of bevacizumab following disease progression after initial bevacizumab treatment and subsequent alternate clinical trial failure. There is a need to conduct further clinical trials to evaluate the benefits of rechallenge with SIACI versus intravenous bevacizumab for GBM and further explore theories of bevacizumab resistance.

Modeling Neisseria meningitidis Infection in Mice: Methods and Logistical Considerations for Nasal Colonization and Invasive Disease.

The single greatest barrier to studying the lifestyle of Neisseria meningitidis stems from its exquisite adaptation to life in humans, a specialization which prevents it from infecting other animals. This barrier to modeling meningococcal infection has been overcome by the provision of factors that allow the meningococci to overcome one or more aspects of host restriction, including the use of mice expressing receptors that allow mucosal colonization and/or the inclusion of serum factors that facilitate meningococcal replication during disseminated meningococcal disease. Here we discuss these advances, consider variables that influence the outcome of infection, and detail the technical requirements to establish robust and reproducible nasal colonization or sepsis. Once established, these models can then be used to study the meningococcal lifestyle and the immune response during infection, and to facilitate development of novel drug or vaccine-based approaches to intervene in meningococcal carriage and disease.

Technical Aspects and Operative Nuances Using a High-Definition 3-Dimensional Exoscope for Cerebral Bypass Surgery.

BACKGROUND: Cerebral bypass operation is a technically challenging operation that requires excellent surgical visibility and efficient ergonomics to minimize complications and maximize successful revascularization. Despite the operative microscope's utilization for the past two generations, there remains a need for continued improvement in operative visualization and surgical ergonomics. OBJECTIVE: To report the positives and negatives of our initial experience using a novel 4 K high-definition (4K-HD) 3-dimensional (3D) exoscope (EX) for cranial bypass surgery. METHODS: A retrospective review over 6 mo was performed of all patients who have undergone cerebral bypass surgery at a single institution using the 4K-HD 3D EX. Advantages and disadvantages of the EX and clinical outcome of the patients were assessed. RESULTS: A total of 5 patients underwent cerebral EC-IC bypass surgery with no EX-related complications and successful revascularization. The lightweight design of the EX allowed for easy instrument maneuverability as well as uncomplicated surgical set up in the operating room. The assistance of the cosurgeon was significantly more efficient compared to that of the operating microscope. The large monitor allowed for an immersive, collaborative, and valuable educational surgical experience. CONCLUSION: Using the EX for cerebral bypass surgery, with 3D ultra-high-definition optics, enhancements of ergonomics, and improved training, we believe that the 3D 4K-HD EX may represent the next generation of operative scopes in microneurosurgery.

Differential influences of complement on neutrophil responses to Neisseria meningitidis infection.

The complement system is the primary innate immune determinant protecting against invasive diseases caused by the Gram-negative bacterium Neisseria meningitidis (Nme, meningococcus), as evidenced by the extreme susceptibility of individuals with complement deficiencies. In contrast, the role of phagocytes such as neutrophils is much less well understood, although they are recruited in great numbers to the cerebrospinal fluid during meningococcal meningitis. Here, we consider the interaction of Nme with primary human neutrophils using either purified cells or a whole blood model of infection. We found that neutrophils are capable of non-opsonic uptake and killing of different Nme strains. However, in the presence of immune serum featuring active complement, Nme association is strongly increased, whereas this is not the case in heat-inactivated immune serum. Blockade of complement at the level of C3 using the inhibitor compstatin Cp20 reduces the uptake dramatically. In addition, purified neutrophils did not mount an oxidative burst towards Nme unless complement was added and, vice versa, the oxidative burst was strongly reduced in whole blood upon complement inhibition. In contrast, there was no significant impact of complement on neutrophil degranulation or IL-8 secretion. Taken together, neutrophils require complement activation in order to mount a full response towards Nme.