The microsporidium Nosema ceranae is a high prevalent parasite of the European honey bee (Apis mellifera). This parasite is spreading across the world into its novel host. The developmental process, and some mechanisms of N. ceranae-infected honey bees, has been studied thoroughly; however, few studies have been carried out in the mechanism of gene expression in N. ceranae during the infection process. We therefore performed the suppressive subtractive hybridization (SSH) approach to investigate the candidate genes of N. ceranae during its infection process. All 96 clones of infected (forward) and non-infected (reverse) library were dipped onto the membrane for hybridization. A total of 112 differentially expressed sequence tags (ESTs) had been sequenced. For the host responses, 20% of ESTs (13 ESTs, 10 genes, and 1 non-coding RNA) from the forward library and 93.6% of ESTs (44 ESTs, 28 genes) from the reverse library were identified as differentially expressed genes (DEGs) of the hosts. A high percentage of DEGs involved in catalytic activity and metabolic processes revealed that the host gene expression change after N. ceranae infection might lead to an unbalance of physiological mechanism. Among the ESTs from the forward library, 75.4% ESTs (49 ESTs belonged to 24 genes) were identified as N. ceranae genes. Out of 24 N. ceranae genes, nine DEGs were subject to real-time quantitative reverse transcription PCR (real-time qRT-PCR) for validation. The results indicated that these genes were highly expressed during N. ceranae infection. Among nine N. ceranae genes, one N. ceranae gene (AAJ76_1600052943) showed the highest expression level after infection. These identified differentially expressed genes from this SSH could provide information about the pathological effects of N. ceranae. Validation of nine up-regulated N. ceranae genes reveal high potential for the detection of early nosemosis in the field and provide insight for further applications.

Antimicrobial resistance (AMR) has important implications for the continued use of antibiotics to control infectious diseases in both beef cattle and humans. AMR along the One Health continuum of the beef production system is largely unknown. Here, whole genomes of presumptive extended-spectrum β-lactamase E. coli (ESBL-EC) from cattle feces (n = 40), feedlot catch basins (n = 42), surrounding streams (n = 21), a beef processing plant (n = 4), municipal sewage (n = 30), and clinical patients (n = 25) are described. ESBL-EC were isolated from ceftriaxone selective plates and subcultured on ampicillin selective plates. Agreement of genotype-phenotype prediction of AMR ranged from 93.2% for ampicillin to 100% for neomycin, trimethoprim/sulfamethoxazole, and enrofloxacin resistance. Overall, β-lactam (100%; blaEC, blaTEM-1, blaSHV, blaOXA, blaCTX-M-), tetracycline (90.1%; tet(A), tet(B)) and folate synthesis (sul2) antimicrobial resistance genes (ARGs) were most prevalent. The ARGs tet(C), tet(M), tet(32), blaCTX-M-1, blaCTX-M-14, blaOXA-1, dfrA18, dfrA19, catB3, and catB4 were exclusive to human sources, while blaTEM-150, blaSHV-11-12, dfrA12, cmlA1, and cmlA5 were exclusive to beef cattle sources. Frequently encountered virulence factors across all sources included adhesion and type II and III secretion systems, while IncFIB(AP001918) and IncFII plasmids were also common. Specificity and prevalence of ARGs between cattle-sourced and human-sourced presumptive ESBL-EC likely reflect differences in antimicrobial use in cattle and humans. Comparative genomics revealed phylogenetically distinct clusters for isolates from human vs. cattle sources, implying that human infections caused by ESBL-EC in this region might not originate from beef production sources.

Most flaviviruses are arthropod-borne viruses, transmitted by either ticks or mosquitoes, and cause morbidity and mortality worldwide. They are endemic in many countries and have recently emerged in new regions, such as the Zika virus (ZIKV) in South-and Central America, the West Nile virus (WNV) in North America, and the Yellow fever virus (YFV) in Brazil and many African countries, highlighting the need for preparedness. Currently, there are no antiviral drugs available to treat flavivirus infections. We have previously discovered a broad-spectrum antiviral compound, benzavir-2, with potent antiviral activity against both DNA- and RNA-viruses. Our purpose was to investigate the inhibitory activity of benzavir-2 against flaviviruses. We used a ZIKV ZsGreen-expressing vector, two lineages of wild-type ZIKV, and other medically important flaviviruses. Benzavir-2 inhibited ZIKV derived reporter gene expression with an EC50 value of 0.8 ± 0.1 µM. Furthermore, ZIKV plaque formation, progeny virus production, and viral RNA expression were strongly inhibited. In addition, 2.5 µM of benzavir-2 reduced infection in vitro in three to five orders of magnitude for five other flaviviruses: WNV, YFV, the tick-borne encephalitis virus, Japanese encephalitis virus, and dengue virus. In conclusion, benzavir-2 was a potent inhibitor of flavivirus infection, which supported the broad-spectrum antiviral activity of benzavir-2.

Atypical teratoid rhabdoid tumors (ATRTs) are among the most malignant brain tumors in early childhood and remain incurable. Myc-ATRT is driven by the Myc oncogene, which directly controls the intracellular protein synthesis rate. Proteasome inhibitor bortezomib (BTZ) was approved by the Food and Drug Administration as a primary treatment for multiple myeloma. This study aimed to determine whether the upregulation of protein synthesis and proteasome degradation in Myc-ATRTs increases tumor cell sensitivity to BTZ. We performed differential gene expression and gene set enrichment analysis on matched primary and recurrent patient-derived xenograft (PDX) samples from an infant with ATRT. Concomitant upregulation of the Myc pathway, protein synthesis and proteasome degradation were identified in recurrent ATRTs. Additionally, we found the proteasome-encoding genes were highly expressed in ATRTs compared with in normal brain tissues, correlated with the malignancy of tumor cells and were essential for tumor cell survival. BTZ inhibited proliferation and induced apoptosis through the accumulation of p53 in three human Myc-ATRT cell lines (PDX-derived tumor cell line Re1-P6, BT-12 and CHLA-266). Furthermore, BTZ inhibited tumor growth and prolonged survival in Myc-ATRT orthotopic xenograft mice. Our findings suggest that BTZ may be a promising targeted therapy for Myc-ATRTs.

Unhealthy Western-style eating patterns (WSEP) predominate, adversely affecting health. Resistance to improving dietary patterns prompts interest to incorporate a potentially health-promoting ingredient into typical WSEP foods and beverages. We assessed the effect of incorporating isocalorically matched carbohydrates versus milk protein isolate (MPI) into a WSEP on weight loss-induced changes in cardiometabolic health and body composition. In a randomized, double-blind, parallel-design study, 44 participants (age 52 ± 1 years, body mass index (BMI) 31.4 ± 0.5 kg/m2, mean ± standard error) consumed a weight maintenance WSEP (0.8 g total protein/kg/day) for 3 weeks (baseline). After, participants consumed an energy-restricted (750 kcal/day below estimated requirement) WSEP for 16 weeks, randomly assigned to contain either an additional 0.7 g carbohydrate/kg/d (CON: n = 23, 0.8 g total protein/kg/day) or 0.7 g protein/kg/d from MPI (MPI: n = 21, 1.5 g total protein/kg/day) incorporated into foods and beverages. Compared to CON, the MPI favored reductions in average 24 h and sleeping systolic and diastolic blood pressures (BP), waking hours systolic BP, and fasting plasma triglyceride concentrations. Reductions in fasting plasma insulin, glucose, total cholesterol, and low-density lipoprotein cholesterol concentrations were not different between groups. Among all participants, whole body mass, lean mass, fat mass, and thigh muscle area, each decreased over time. For adults finding it difficult to deviate from a WSEP, replacing a portion of their carbohydrate with foods and beverages containing MPI may be an effective dietary strategy to reduce BP after weight loss.

Natural products play vital roles against infectious diseases since ancient times and most drugs in use today are derived from natural sources. Worldwide, multi-drug resistance becomes a massive threat to the society with increasing mortality. Hence, it is very crucial to identify alternate strategies to control these 'super bugs'. Pseudomonas aeruginosa is an opportunistic pathogen reported to be resistant to a large number of critically important antibiotics. Quorum sensing (QS) is a cell-cell communication mechanism, regulates the biofilm formation and virulence factors that endow pathogenesis in various bacteria including P. aeruginosa. In this study, we identified and evaluated quorum sensing inhibitors (QSIs) from plant-based natural products against P. aeruginosa. In silico studies revealed that catechin-7-xyloside (C7X), sappanol and butein were capable of interacting with LasR, a LuxR-type quorum sensing regulator of P. aeruginosa. In vitro assays suggested that these QSIs significantly reduced the biofilm formation, pyocyanin, elastase, and rhamnolipid without influencing the growth. Especially, butein reduced the biofilm formation up to 72.45% at 100 µM concentration while C7X and sappanol inhibited the biofilm up to 66% and 54.26% respectively. Microscale thermophoresis analysis revealed that C7X had potential interaction with LasR (KD = 933±369 nM) and thermal shift assay further confirmed the biomolecular interactions. These results suggested that QSIs are able to substantially obstruct the P. aeruginosa QS. Since LuxR-type transcriptional regulator homologues are present in numerous bacterial species, these QSIs may be developed as broad spectrum anti-infectives in the future.

This study designed a vertical take-off and landing tailsitter unmanned aerial vehicle (UAV) with a long endurance time. Nine parameters of the tailsitter UAV were investigated. Using a 2k full factorial test, 512 experiments on the nine parameters were conducted at their maximum and minimum values. The time coefficient and air resistance were calculated using the computational fluid dynamics (CFD) method under different parameter combinations. The analysis of variance determined that the specific factors influencing the time coefficient and air resistance were the root chord, wingtip chord, wingspan, and sweep angle. By carrying out a central composite design (CCD) test, 25 sample points of the four particular factors were constructed. The time coefficient and air resistance were simulated under different structural parameter combinations using the CFD method. CFD simulation was verified by carrying out a wind tunnel test, and the results revealed that the aerodynamic coefficient error was less than 5%, while the air resistance error was less than 6%. The response surface methodology (RSM) for the time coefficient and air resistance was established using a genetic aggregation method. A multi-objective genetic algorithm (MOGA) was used to optimize the parameters with regard to the maximum time coefficient and minimum air resistance. The optimal structural parameters were wing root chord length at 315 mm, wingtip chord length at 182 mm, wingspan length at 1198 mm, and sweep angle at 16°. Compared with the original layout and size, the time coefficient of the new design of the tailsitter UAV improved by 19.5%, while the air resistance reduced by 34.78%. The results obtained by this study are significant for the design of tailsitter UAVs.

Cell-penetrating peptides might have great potential for enhancing the therapeutic effect of drug molecules against such dangerous pathogens as Mycobacterium tuberculosis (Mtb), which causes a major health problem worldwide. A set of cationic cell-penetration peptides with various hydrophobicity were selected and synthesized as drug carrier of isoniazid (INH), a first-line antibacterial agent against tuberculosis. Molecular interactions between the peptides and their INH-conjugates with cell-membrane-forming lipid layers composed of DPPC and mycolic acid (a characteristic component of Mtb cell wall) were evaluated, using the Langmuir balance technique. Secondary structure of the INH conjugates was analyzed and compared to that of the native peptides by circular dichroism spectroscopic experiments performed in aqueous and membrane mimetic environment. A correlation was found between the conjugation induced conformational and membrane affinity changes of the INH-peptide conjugates. The degree and mode of interaction were also characterized by AFM imaging of penetrated lipid layers. In vitro biological evaluation was performed with Penetratin and Transportan conjugates. Results showed similar internalization rate into EBC-1 human squamous cell carcinoma, but markedly different subcellular localization and activity on intracellular Mtb.

A simple and inexpensive method using planar electrodes was proposed for the measurement of the imaginary part of the Clausius-Mossotti factor, K i , of particle/cell for electrorotation (ER) and travelling wave dielectrophoresis (twDEP). It is based on the balance between the dielectrophoretic and viscous torques on a particle undergoing ER subject to dual frequency operation in an ER chamber. A four-phase ac voltage signal with a given frequency is applied for generating ER for measurement, and another two-phase signal is applied at a selected frequency for generating a negative dielectrophoretic force for confining the particle motion, instead of using laser tweezer or three-dimensional electrodes in the literature. Both frequencies can be applied to the same electrodes in a four-electrode ER system and to alternative different electrodes in an eight-electrode ER system, and both systems are capable for providing accurate measurement. The measurements were validated by comparing with the theoretical result using sephadex particles in KCl solution, and with the existing experimental results for various human cancer cells in medium with conductivity from 0.01-1.2 S/m, using ER with optical tweezer and dual frequency twDEP. Contrast between the ER and the twDEP methods (the current two available methods) was discussed and commented. The present method could provide measurement for wider frequency range and more accurate result near K i = 0, in comparison with the results using the twDEP method. However, the twDEP method could perform much more rapid measurement. Detailed forces and torque were calculated inside the ER chamber for understanding the physics and assessing the characteristics of the dual frequency ER method. This study is of academic interest as the torque in ER and the force in twDEP can be calculated only when K i is known. It also finds biomedical applications as the K i -spectra can be served as physical phenotypes for different cells, and can be applied for deriving dielectric properties of cells.

Diabetic retinopathy (DR), Retinopathy of Pre-maturity (ROP), and Age-related Macular Degeneration (AMD) are multifactorial manifestations associated with abnormal growth of blood vessels in the retina. These three diseases account for 5% of the total blindness and vision impairment in the US alone. The current treatment options involve heavily invasive techniques such as frequent intravitreal administration of anti-VEGF (vascular endothelial growth factor) antibodies, which pose serious risks of endophthalmitis, retinal detachment and a multitude of adverse effects stemming from the diverse physiological processes that involve VEGF. To overcome these limitations, this current study utilizes a micellar delivery vehicle (MC) decorated with an anti-angiogenic peptide (aANGP) that inhibits αvβ3 mediated neovascularization using primary endothelial cells (HUVEC). Stable incorporation of the peptide into the micelles (aANGP-MCs) for high valency surface display was achieved with a lipidated peptide construct. After 24 h of treatment, aANGP-MCs showed significantly higher inhibition of proliferation and migration compared to free from aANGP peptide. A tube formation assay clearly demonstrated a dose-dependent angiogenic inhibitory effect of aANGP-MCs with a maximum inhibition at 4 μg/mL, a 1000-fold lower concentration than that required for free from aANGP to display a biological effect. These results demonstrate valency-dependent enhancement in the therapeutic efficacy of a bioactive peptide following conjugation to nanoparticle surfaces and present a possible treatment alternative to anti-VEGF antibody therapy with decreased side effects and more versatile options for controlled delivery.

Vitamin D is associated with cardiovascular health through activating the vitamin D receptor that targets genes related to cardiovascular disease (CVD). The human cardiac microvascular endothelial cells (HCMECs) were used to develop mechanically and TGF-β1-induced fibrosis models, and the rat was used as the isoproterenol (ISO)-induced fibrosis model. The rats were injected with ISO for the first five days, followed by vitamin D injection for the consecutive three weeks before being sacrificed on the fourth week. Results showed that mechanical stretching reduced endothelial cell marker CD31 and VE-cadherin protein expressions, as well as increased α-smooth muscle actin (α-SMA) and fibronectin (FN). The transforming growth factor-β1 (TGF-β1) reduced CD31, and increased α-SMA and FN protein expression levels. Vitamin D presence led to higher protein expression of CD31, and lower protein expressions of α-SMA and FN compared to the control in the TGF-β1-induced fibrosis model. Additionally, protein expression of VE-cadherin was increased and fibroblast-specific protein-1 (FSP1) was decreased after vitamin D treatment in the ISO-induced fibrosis rat. In conclusion, vitamin D slightly inhibited fibrosis development in cell and animal models. Based on this study, the beneficial effect of vitamin D may be insignificant; however, further investigation of vitamin D's effect in the long-term is required in the future.

Non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in adults and children. It is characterized by excessive accumulation of lipids in the hepatocytes of patients without any excess alcohol intake. With a global presence of 24% and limited therapeutic options, the disease burden of NAFLD is increasing. Thus, it becomes imperative to attempt to understand the dynamics of disease progression at a systems-level. Here, we decoded the emergent dynamics of underlying gene regulatory networks that were identified to drive the initiation and the progression of NAFLD. We developed a mathematical model to elucidate the dynamics of the HNF4α-PPARγ gene regulatory network. Our simulations reveal that this network can enable multiple co-existing phenotypes under certain biological conditions: an adipocyte, a hepatocyte, and a "hybrid" adipocyte-like state of the hepatocyte. These phenotypes may also switch among each other, thus enabling phenotypic plasticity and consequently leading to simultaneous deregulation of the levels of molecules that maintain a hepatic identity and/or facilitate a partial or complete acquisition of adipocytic traits. These predicted trends are supported by the analysis of clinical data, further substantiating the putative role of phenotypic plasticity in driving NAFLD. Our results unravel how the emergent dynamics of underlying regulatory networks can promote phenotypic plasticity, thereby propelling the clinically observed changes in gene expression often associated with NAFLD.

The pathogenicity of the common phytopathogenic bacterium Pseudomonas syringae toward Caenorhabditis elegans has been recently demonstrated. However, the major virulence factors involved in this interaction remain unknown. In this study, we investigated the nematocidal activity of P. syringae against C. elegans under iron-sufficient/limited conditions, primarily focusing on the role of the ferric chelator pyoverdine in a P. syringae-C. elegans liquid-based pathogenicity model. Prediction-based analysis of pyoverdine-encoding genes in the genome of the wild-type P. syringae strain MB03 revealed that the genes are located in one large cluster. Two non-ribosomal peptide synthetase genes (pvdD and pvdJ) were disrupted via a Rec/TE recombination system, resulting in mutant strains with abrogated pyoverdine production and attenuated virulence against C. elegans. When used alone, pure pyoverdine also showed nematocidal activity. The role of iron used alone or with pyoverdine was further investigated in mutant and MB03-based bioassays. The results indicated that pyoverdine in P. syringae MB03 is a robust virulence factor that promotes the killing of C. elegans. We speculate that pyoverdine functions as a virulence determinant by capturing environmentally available iron for host bacterial cells, by limiting its availability for C. elegans worms, and by regulating and/or activating other intracellular virulence factors that ultimately kills C. elegans worms.

Nearly half of all cancers are treated with radiotherapy alone or in combination with other treatments, where damage to normal tissues is a limiting factor for the treatment. Radiotherapy-induced adverse health effects, mostly of importance for cancer patients with long-term survival, may appear during or long time after finishing radiotherapy and depend on the patient's radiosensitivity. Currently, there is no assay available that can reliably predict the individual's response to radiotherapy. We profiled two study sets from breast (n = 29) and head-and-neck cancer patients (n = 74) that included radiosensitive patients and matched radioresistant controls.. We studied 55 single nucleotide polymorphisms (SNPs) in 33 genes by DNA genotyping and 130 circulating proteins by affinity-based plasma proteomics. In both study sets, we discovered several plasma proteins with the predictive power to find radiosensitive patients (adjusted p < 0.05) and validated the two most predictive proteins (THPO and STIM1) by sandwich immunoassays. By integrating genotypic and proteomic data into an analysis model, it was found that the proteins CHIT1, PDGFB, PNKD, RP2, SERPINC1, SLC4A, STIM1, and THPO, as well as the VEGFA gene variant rs69947, predicted radiosensitivity of our breast cancer (AUC = 0.76) and head-and-neck cancer (AUC = 0.89) patients. In conclusion, circulating proteins and a SNP variant of VEGFA suggest that processes such as vascular growth capacity, immune response, DNA repair and oxidative stress/hypoxia may be involved in an individual's risk of experiencing radiation-induced toxicity.

CD1 molecules present lipid antigens for recognition by T-cell receptors (TCRs). Although a reasonably detailed picture of the CD1-lipid-TCR interaction exists, the initial steps regarding lipid loading onto and exchange between CD1 proteins remain elusive. The hydrophobic nature of lipids and the fact that CD1 molecules are unable to extract lipids from membranes raise the need for the assistance of helper proteins in lipid trafficking. However, the experimental study of this traffic in the endosomal compartments at which it occurs is so challenging that computational studies can help provide mechanistic insight into the associated processes. Here we present a multifaceted computational approach to obtain dynamic structural data on the human CD1d isotype. Conformational dynamics analysis shows an intrinsic flexibility associated with the protein architecture. Electrostatic properties together with molecular dynamics results for CD1d complexes with several lipids and helper proteins unravel the high dynamic plasticity of the antigen-binding site that is crucially favoured by acidic pH and the presence of helper proteins.

Imidacloprid (IMI) is one of the most frequently used neonicotinoid insecticides, but recent studies have shown adverse effects on mammals. IMI was found to be neurotoxic and hepatotoxic. In the present study, the effects of repeated oral administration of two doses of IMI (5 and 20 mg/kg/day) for 28 days on hippocampus and liver of female KM mice were studied. The histopathological and biochemical experiments indicated obvious damages to the hippocampus and liver of mice in the high-dose group (20 mg/kg/day). Using a high-throughput metabolomics platform based on ultrahigh performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS), we studied effects of IMI on metabolic profiles in the hippocampus and liver of mice. Significant differences among the control group, the low-dose group and the high-dose group were clearly presented using multivariate analysis. The changed metabolic profile in the low-dose group (5 mg/kg/day) revealed that the metabolic disturbance in the hippocampus and liver of mice had been induced by low-dose of IMI, although no significant histopathological changes were observed in the low-dose group. Six differential metabolites in the hippocampus and 10 differential metabolites in the liver were identified as the possible biomarkers to distinguish IMI exposure from the control group using the variable importance in projection (VIP) value and receiver operating characteristic (ROC) analysis. The metabolism disturbances of important biochemical pathways in the hippocampus and liver of mice in the exposed groups were elucidated, mostly concentrated in lipid metabolism, amino acid metabolism, nucleotide metabolism, carbohydrate metabolism, and energy metabolism (p < 0.05). Such investigations give out a global view of IMI-induced damages in the hippocampus and liver of mice and imply a health risk associated with early metabolic damage in mice.

The genetically heterogeneous triple-negative breast cancer (TNBC) continues to be an intractable disease, due to lack of effective targeted therapies. Gene amplification is a major event in tumorigenesis. Genes with amplification-dependent expression are being explored as therapeutic targets for cancer treatment. In this study, we have applied Analytical Multi-scale Identification of Recurring Events analysis and transcript quantification in the TNBC genome across 222 TNBC tumors and identified 138 candidate genes with positive correlation in copy number gain (CNG) and gene expression. siRNA-based loss-of-function screen of the candidate genes has validated EGFR, MYC, ASAP1, IRF2BP2, and CCT5 genes as drivers promoting proliferation in different TNBC cells. MYC, ASAP1, IRF2BP2, and CCT5 display frequent CNG and concurrent expression over 2173 breast cancer tumors (cBioPortal dataset). More frequently are MYC and ASAP1 amplified in TNBC tumors (>30%, n = 320). In particular, high expression of ASAP1, the ADP-ribosylation factor GTPase-activating protein, is significantly related to poor metastatic relapse-free survival of TNBC patients (n = 257, bc-GenExMiner). Furthermore, we have revealed that silencing of ASAP1 modulates numerous cytokine and apoptosis signaling components, such as IL1B, TRAF1, AIFM2, and MAP3K11 that are clinically relevant to survival outcomes of TNBC patients. ASAP1 has been reported to promote invasion and metastasis in various cancer cells. Our findings that ASAP1 is an amplification-dependent TNBC driver gene promoting TNBC cell proliferation, functioning upstream apoptosis components, and correlating to clinical outcomes of TNBC patients, support ASAP1 as a potential actionable target for TNBC treatment.

Toll-like receptor (TLR) signaling pathways need to be tightly controlled to avoid excessive inflammation and unwanted damage to the host. Myeloid differentiation primary response gene 88 (MyD88) is a critical adaptor of TLR signaling. Here, we identified the speckle-type POZ protein (SPOP) as a MyD88-associated protein. SPOP was recruited to MyD88 following TLR4 activation. TLR4 activation also caused the translocation of SPOP from the nucleus to the cytoplasm. SPOP depletion promoted the aggregation of MyD88 and recruitment of the downstream signaling kinases IRAK4, IRAK1 and IRAK2. Consistently, overexpression of SPOP inhibited the TLR4-mediated activation of NF-κB and production of inflammatory cytokines, whereas SPOP depletion had the opposite effects. Furthermore, knockdown of SPOP increased MyD88 aggregation and inflammatory cytokine production upon TLR2, TLR7 and TLR9 activation. Our findings reveal a mechanism by which MyD88 is regulated and highlight a role for SPOP in limiting inflammatory responses.

Induction of broadly neutralizing monoclonal antibodies (bNAbs) that bind to the viral envelope glycoproteins is a major goal of hepatitis C virus (HCV) vaccine research. The study of bNAbs arising in natural infection is essential in this endeavor. We generated a human antibody, 8D6, recognizing the E2 protein of HCV isolated from a chronic hepatitis C patient. This antibody shows broadly neutralizing activity, which covers a pan-genotypic panel of cell culture-derived HCV virions (HCVcc). Functional and epitope analyses demonstrated that 8D6 can block the interaction between E2 and CD81 by targeting a highly conserved epitope on E2. We describe how the 8D6 lineage evolved via somatic hypermutation to achieve broad neutralization. We found that the V(D)J recombination-generated junctional and somatic hypermutation-induced disulfide bridge (C-C) motif in the CDRH3 is critical for the broad neutralization and binding activity of 8D6. This motif is conserved among a series of broadly neutralizing HCV antibodies, indicating a common binding model. Next, the 8D6 inferred germline (iGL) was reconstructed and tested for its binding affinity and neutralization activity. Interestingly, 8D6 iGL-mediated relatively strong inhibition of the 1b genotype PR79L9 strain, suggesting that PR79L9 may serve as a potential natural viral strain that provides E2 sequences that induce bNAbs. Overall, our detailed epitope mapping and genetic studies of the HCV E2-specific mAb 8D6 have allowed for further refinement of antigenic sites on E2 and reveal a new mechanism to generate a functional CDRH3, while its iGL can serve as a probe to identify potential HCV vaccine strains.

When used appropriately, a confocal fluorescence microscope is an excellent tool for making quantitative measurements in cells and tissues. The confocal microscope's ability to block out-of-focus light and thereby perform optical sectioning through a specimen allows the researcher to quantify fluorescence with very high spatial precision. However, generating meaningful data using confocal microscopy requires careful planning and a thorough understanding of the technique. In this tutorial, the researcher is guided through all aspects of acquiring quantitative confocal microscopy images, including optimizing sample preparation for fixed and live cells, choosing the most suitable microscope for a given application and configuring the microscope parameters. Suggestions are offered for planning unbiased and rigorous confocal microscope experiments. Common pitfalls such as photobleaching and cross-talk are addressed, as well as several troubling instrumentation problems that may prevent the acquisition of quantitative data. Finally, guidelines for analyzing and presenting confocal images in a way that maintains the quantitative nature of the data are presented, and statistical analysis is discussed. A visual summary of this tutorial is available as a poster (https://doi.org/10.1038/s41596-020-0307-7).