Pain Prevalence and Management in a General Hospital Through Repeated Cross-Sectional Surveys in 2011 and 2021.

Background: There is great scope for improving the quality of pain management. Although pain prevalence has been investigated in several countries, few studies have comparatively assessed changes in pain prevalence and management over a span of multiple years. Aim: This work was aimed at determining the pain prevalence and evaluating the condition of pain management in a Chinese general hospital in 2021 and comparing them with corresponding data from 10 years ago. Methods: Repeated single-center cross-sectional studies were initiated on June 14th, 2011, and September 2nd, 2021, in the same tertiary grade A Chinese general hospital. The same structured questionnaire was used to collect inpatient data on pain intensity and classification and pain management outcomes. We performed statistical analyses to compare categorical variables to assess changes over time. Results: The sample sizes for the investigations in 2011 and 2021 were 2323 and 4454, respectively. In 2021, 24.34% of patients experienced pain; this percentage was significantly lower than that in 2011. Meanwhile, the prevalence of moderate and severe pain decreased from 14.73% in 2011 to 4.98% in 2021. The other six indicators of pain management outcomes also improved significantly. The percentages of patients using painkillers, opioid analgesics, and multiple analgesics increased from 44.61 to 51.38%, 24.01% to 44.61%, and 6.82% to 14.11%, respectively. Furthermore, the percentages of patients who received pain information and who actively reported pain increased from 27.56% to 96.5% and from 85.54% to 98.71%, respectively. The percentage of patients qualified to accurately use the Numerical Rating Scale increased from 10.5% to 79.98%. Conclusion: The quality and outcomes of pain management improved greatly after the establishment and implementation of the pain management system. Nonetheless, pain of different intensities is common after major surgeries, and it is recommended that hospitals popularize and implement perioperative multimodal analgesia strategies to reduce the incidence of postoperative pain.

Function-based high-throughput screening for antibody antagonists and agonists against G protein-coupled receptors.

Hybridoma and phage display are two powerful technologies for isolating target-specific monoclonal antibodies based on the binding. However, for complex membrane proteins, such as G protein-coupled receptors (GPCRs), binding-based screening rarely results in functional antibodies. Here we describe a function-based high-throughput screening method for quickly identifying antibody antagonists and agonists against GPCRs by combining glycosylphosphatidylinositol-anchored antibody cell display with ß-arrestin recruitment-based cell sorting and screening. This method links antibody genotype with phenotype and is applicable to all GPCR targets. We validated this method by identifying a panel of antibody antagonists and an antibody agonist to the human apelin receptor from an immune antibody repertoire. In contrast, we obtained only neutral binders and antibody antagonists from the same repertoire by phage display, suggesting that the new approach described here is more efficient than traditional methods in isolating functional antibodies. This new method may create a new paradigm in antibody drug discovery.

Structure-guided discovery of a single-domain antibody agonist against human apelin receptor.

Developing antibody agonists targeting the human apelin receptor (APJ) is a promising therapeutic approach for the treatment of chronic heart failure. Here, we report the structure-guided discovery of a single-domain antibody (sdAb) agonist JN241-9, based on the cocrystal structure of APJ with an sdAb antagonist JN241, the first cocrystal structure of a class A G protein-coupled receptor (GPCR) with a functional antibody. As revealed by the structure, JN241 binds to the extracellular side of APJ, makes critical contacts with the second extracellular loop, and inserts the CDR3 into the ligand-binding pocket. We converted JN241 into a full agonist JN241-9 by inserting a tyrosine into the CDR3. Modeling and molecular dynamics simulation shed light on JN241-9-stimulated receptor activation, providing structural insights for finding agonistic antibodies against class A GPCRs.

Using the Delphi method to establish nursing-sensitive quality indicators for ICU nursing in China.

In this study, the Delphi method was used to develop evidence-based indicators of intensive care unit (ICU) nursing quality of care in China. Nursing quality indicators reflect elements of patient care that are directly affected by nursing practice. A comprehensive literature search identified 2,857 potentially relevant articles. From the 50 articles that were included in this study, researchers identified 38 commonly used nursing quality indicators. A panel of experts reduced these to 20, which were then subjected to two rounds of Delphi discussion by a different panel, and a final consensus was achieved. The 20 indicators were grouped into three dimensions: structure, process, and outcome (including adverse consequences). The agreement among the experts for the 20 indicators was high. These evidence-based nursing quality indicators provide for ease in data collection and a basis for clinical application and improvement in the quality of ICU nursing throughout China.

Long-read sequencing identified intronic repeat expansions in SAMD12 from Chinese pedigrees affected with familial cortical myoclonic tremor with epilepsy.

BACKGROUND: The locus for familial cortical myoclonic tremor with epilepsy (FCMTE) has long been mapped to 8q24 in linkage studies, but the causative mutations remain unclear. Recently, expansions of intronic TTTCA and TTTTA repeat motifs within SAMD12 were found to be involved in the pathogenesis of FCMTE in Japanese pedigrees. We aim to identify the causative mutations of FCMTE in Chinese pedigrees. METHODS: We performed genetic linkage analysis by microsatellite markers in a five-generation Chinese pedigree with 55 members. We also used array-comparative genomic hybridisation (CGH) and next-generation sequencing (NGS) technologies (whole-exome sequencing, capture region deep sequencing and whole-genome sequencing) to identify the causative mutations in the disease locus. Recently, we used low-coverage (~10×) long-read genome sequencing (LRS) on the PacBio Sequel and Oxford Nanopore platforms to identify the causative mutations, and used repeat-primed PCR for validation of the repeat expansions. RESULTS: Linkage analysis mapped the disease locus to 8q23.3-24.23. Array-CGH and NGS failed to identify causative mutations in this locus. LRS identified the intronic TTTCA and TTTTA repeat expansions in SAMD12 as the causative mutations, thus corroborating the recently published results in Japanese pedigrees. CONCLUSIONS: We identified the pentanucleotide repeat expansion in SAMD12 as the causative mutation in Chinese FCMTE pedigrees. Our study also suggested that LRS is an effective tool for molecular diagnosis of genetic disorders, especially for neurological diseases that cannot be positively diagnosed by conventional clinical microarray and NGS technologies.

GPCR structure and function relationship: identification of a biased apelin receptor mutant.

Biased ligands of G protein-coupled receptors (GPCRs) may have improved therapeutic benefits and safety profiles. However, the molecular mechanism of GPCR biased signaling remains largely unknown. Using apelin receptor (APJ) as a model, we systematically investigated the potential effects of amino acid residues around the orthosteric binding site on biased signaling. We discovered that a single residue mutation I109A (I1093.32) in the transmembrane domain 3 (TM3) located in the deep ligand-binding pocket was sufficient to convert a balanced APJ into a G protein signaling biased receptor. APJ I109A mutant receptor retained full capabilities in ligand binding and G protein activation, but was defective in GRK recruitment, ß-arrestin recruitment, and downstream receptor-mediated ERK activation. Based on molecular dynamics simulations, we proposed a molecular mechanism for biased signaling of I109A mutant receptor. We postulate that due to the extra space created by I109A mutation, the phenyl group of the last residue (Phe-13) of apelin rotates down and initiates a cascade of conformational changes in TM3. Phe-13 formed a new cluster of hydrophobic interactions with the sidechains of residues in TM3, including F1103.33 and M1133.36, which stabilizes the mutant receptor in a conformation favoring biased signaling. Interruption of these stabilizing interactions by double mutation F110A/I109A or M113A/I109A largely restored the ß-arrestin-mediated signaling. Taken together, we describe herein the discovery of a biased APJ mutant receptor and provide detailed molecular insights into APJ signaling selectivity, facilitating the discovery of novel therapeutics targeting APJ.

Development of evidence-based nursing-sensitive quality indicators for emergency nursing: A Delphi study.

AIMS AND OBJECTIVES: To establish evidence-based nursing-sensitive quality indicators for emergency nursing in China. BACKGROUND: China lacks nursing-sensitive quality indicators necessary for assessing the quality of emergency nursing and essential to nursing management. DESIGN: Prospective. METHODS: A literature search for relevant evidence-based studies was performed using several databases from January 2009-May 2014. Previously reported quality indicators were identified as appropriate for assessment by a panel of 40 experts in emergency medicine and nursing. Two successive rounds of Delphi surveys were conducted using questionnaires designed by the experts. Kendal's W coordination coefficients were calculated for indicator importance, rationality of calculation and feasibility of data collection. RESULTS: Thirty-three quality indicators were initially proposed for expert evaluation. After round 1 of expert discussion, Kendal's W coordination coefficients were .152 for importance, .092 for rationality and .141 for feasibility of data collection (all p < .001). Seven unsuitable items were discarded in round 1 and 11 discarded in round 2, which also added one new item. Finally, the experts reached consensus on 16 items established as appropriate nursing-sensitive quality indicators for emergency nursing care. CONCLUSION: Evidence-based nursing-sensitive quality indicators were established through a consensus of experts in emergency nursing and medicine. RELEVANCE TO CLINICAL PRACTICE: The current findings may provide a theoretical basis for establishing an emergency nursing quality database and improving the quality of emergency nursing care in China.

Construction of a recombinant full-length membrane associated IgG library.

HIV/AIDS has become a global pandemic. Development of an effective HIV-1 vaccine eliciting broadly neutralizing monoclonal antibodies (bnmAbs) remains a big challenge. Before an effective vaccine comes out, passive treatment for prevention and protection of HIV-1 infection may alleviate the burden caused by the pandemic. Numerous bnmAbs have been isolated against different epitopes in HIV-1 envelope glycoprotein via phage/yeast display, EBV-immortalization, single cell sorting and micro neutralization. Recombinant antibody library with extended diversity and enlarged size of units are applicable by phage/yeast display and mammalian cell display for monoclonal antibody isolation. Here, we constructed an immune recombinant membrane associated full length IgGs library based on mammalian cell display system. This library can be used for monoclonal antibody screening/isolation by target cell sorting. A full length antibody mz2F11 was screened with potent neutralizing activities against a panel of viruses tested. Our study provides a novel way of antibody library construction and monoclonal antibody screening. Antibodies screened via this method can help broaden the knowledge in passive treatment and prevention against HIV-1 infection.

Antibody-Dependent Cell-Mediated Cytotoxicity Epitopes on the Hemagglutinin Head Region of Pandemic H1N1 Influenza Virus Play Detrimental Roles in H1N1-Infected Mice.

Engaging the antibody-dependent cell-mediated cytotoxicity (ADCC) for killing of virus-infected cells and secretion of antiviral cytokines and chemokines was incorporated as one of the important features in the design of universal influenza vaccines. However, investigation of the ADCC epitopes on the highly immunogenic influenza hemagglutinin (HA) head region has been rarely reported. In this study, we determined the ADCC and antiviral activities of two putative ADCC epitopes, designated E1 and E2, on the HA head of a pandemic H1N1 influenza virus in vitro and in a lethal mouse model. Our data demonstrated that sera from the E1-vaccinated mice could induce high ADCC activities. Importantly, the induction of ADCC response modestly decreased viral load in the lungs of H1N1-infected mice. However, the elevated ADCC significantly increased mouse alveolar damage and mortality than that of the PBS-vaccinated group (P < 0.0001). The phenotype was potentially due to an exaggerated inflammatory cell infiltration triggered by ADCC, as an upregulated release of cytotoxic granules (perforin) was observed in the lung tissue of E1-vaccinated mice after H1N1 influenza virus challenge. Overall, our data suggested that ADCC elicited by certain domains of HA head region might have a detrimental rather than protective effect during influenza virus infection. Thus, future design of universal influenza vaccine shall strike a balance between the induction of protective immunity and potential side effects of ADCC.

Using the Delphi method to develop nursing-sensitive quality indicators for the NICU.

AIMS AND OBJECTIVES: To develop nursing-sensitive quality indicators consistent with current medical practices in Chinese neonatal intensive care units. BACKGROUND: The development of nursing-sensitive quality indicators has become a top priority in nursing management. To the best of our knowledge, there has been no objective, scientific and sensitive evaluation of the quality of neonatal intensive care unit nursing in China. DESIGN: A modified Delphi technique was used to seek opinions from experts about what should be used and prioritised as indicators of quality care in neonatal intensive care unit nursing. METHODS: Based on a literature review, we identified 21 indicators of nursing-sensitive quality in the neonatal intensive care unit. Our group of 11 consultants chose 13 indicators to be discussed using the Delphi method. In October and November 2014, 39 neonatal intensive care unit experts in 18 tertiary hospitals spread across six provinces participated in two rounds of Delphi panels. RESULTS: Of the 13 indicators discussed, 11 were identified as indicators of nursing-sensitive quality in the neonatal intensive care unit: rate of nosocomial infections, rate of accidental endotracheal extubation, rate of errors in medication administration, rate of treatment for pain, rate of peripheral venous extravasation, rate of compliance with handwashing techniques, incidence of pressure ulcers, incidence of noise, the bed-to-care ratio, the proportion of nurses with greater than five years neonatal intensive care unit experience and incidence of retinopathy. CONCLUSIONS: The 11 neonatal intensive care unit nursing-sensitive indicators identified by the Delphi method integrated with basic Chinese practices provide a basis for nursing management and the monitoring of nursing quality. RELEVANCE TO CLINICAL PRACTICE: This study identified nursing-sensitive quality indicators for neonatal intensive care unit care that are suitable for current clinical practice in China.

Identification of Human Anti-HIV gp160 Monoclonal Antibodies That Make Effective Immunotoxins.

The envelope (Env) glycoprotein of HIV is the only intact viral protein expressed on the surface of both virions and infected cells. Env is the target of neutralizing antibodies (Abs) and has been the subject of intense study in efforts to produce HIV vaccines. Therapeutic anti-Env Abs can also exert antiviral effects via Fc-mediated effector mechanisms or as cytotoxic immunoconjugates, such as immunotoxins (ITs). In the course of screening monoclonal antibodies (MAbs) for their ability to deliver cytotoxic agents to infected or Env-transfected cells, we noted disparities in their functional activities. Different MAbs showed diverse functions that did not correlate with each other. For example, MAbs against the external loop region of gp41 made the most effective ITs against infected cells but did not neutralize virus and bound only moderately to the same cells that they killed so effectively when they were used in ITs. There were also differences in IT-mediated killing among transfected and infected cell lines that were unrelated to the binding of the MAb to the target cells. Our studies of a well-characterized antigen demonstrate that MAbs against different epitopes have different functional activities and that the binding of one MAb can influence the interaction of other MAbs that bind elsewhere on the antigen. These results have implications for the use of MAbs and ITs to kill HIV-infected cells and eradicate persistent reservoirs of HIV infection. IMPORTANCE: There is increased interest in using antibodies to treat and cure HIV infection. Antibodies can neutralize free virus and kill cells already carrying the virus. The virus envelope (Env) is the only HIV protein expressed on the surfaces of virions and infected cells. In this study, we examined a panel of human anti-Env antibodies for their ability to deliver cell-killing toxins to HIV-infected cells and to perform other antiviral functions. The ability of an antibody to make an effective immunotoxin could not be predicted from its other functional characteristics, such as its neutralizing activity. Anti-HIV immunotoxins could be used to eliminate virus reservoirs that persist despite effective antiretroviral therapy.

Establishing nursing-sensitive quality indicators for the operating room: A cross-sectional Delphi survey conducted in China.

BACKGROUND: Nursing-sensitive quality indicators comprise principles, procedures, and assessments to quantify the level of nursing quality in hospital departments. Although studies have demonstrated that quality indicators are essential for monitoring nursing practice in the operating room (OR), nursing quality in China is highly subjective and localised OR nursing-sensitive quality indicators are lacking. OBJECTIVE: This study aimed to establish scientific, objective and comprehensive nursing-sensitive quality indicators for the OR to evaluate and monitor OR nursing care quality in China. METHODS: Literature search for relevant evidence-based studies was performed using Cochrane, Medline, PubMed, Embase, and other databases, followed by literature review and group discussion by the expert panel. Two successive rounds of Delphi surveys were conducted using questionnaires completed by the expert panel to reach consensus and define nursing-sensitive quality indicators for the OR. RESULTS: Two rounds of Delphi surveys each had 100% questionnaire retrieval rate, with Kendall W coordination coefficients ranging from 0.096 to 0.263 (P<0.001). In round 1 of expert evaluation of 26 indicators, Kendall's W was 0.263 for importance, 0.126 for rationality, and 0.125 for feasibility of data collection (all P<0.001). After round 2, 23 items were established as OR nursing-sensitive quality indicators, including rates of work time wastage, surgery start-time delay, OR turnover time between surgeries, same-day surgery cancellation, and number of monthly surgeries in each OR; checking surgical patients, surgery site marking, allergy history, and antibiotics use 60min before incision; and also assessing expected surgical time, sterilisation indicator results, availability of surgical instruments and materials, and instrument count. CONCLUSIONS: Scientific, practical, and reliable OR nursing-sensitive quality indicators can be established based on evidence-based studies and expert consensus using the Delphi method. The quality indicators developed in this study may provide an objective and quantitative reference for evaluating nursing quality in Chinese ORs.

Correlation study of Bcl-2, B7-H1, EGFR, VEGF and colorectal cancer.

OBJECTIVE: To analyze the expressions of Bcl-2, B7-H1, EGFR and VEGF in colorectal cancer for the further investigation of their correlations with the clinical pathological features of colorectal cancer. METHOD: Fresh colorectal cancer tissues and the expressions of Bcl-2, B7-H1, VEGF and EGFR in paraneoplastic normal mucosal tissues of 57 cases were tested by immunohistochemisty method, and the results were analyzed by SPSS10.0. RESULTS: 1. Compared with paraneoplastic normal tissues, the expressions of Bcl-2 and B7-H1 in colorectal cancer tissues increased significantly with significant difference (P<0.05), while the expression of EGFR and VEGF in colorectal cancer tissues showed no significant difference with those in paraneoplastic normal tissue (P>0.05); 2. The correlation with clinical pathological features: there was significant difference of expression rates of EGFR between different genders (P<0.05); the expressions of BCL-2 and B7-H1 in colorectal cancer of the high- and medium- differentiated groups were significantly higher than those of the low-differentiated group, and the difference was significant (P<0.01); compared with the colorectal cancer patients without lymph node metastasis (Dukes stage A+B), the expression of B7-H1 in patients with lymph node metastasis (Dukes stage C+D) was significantly higher (P<0.05); 3. Within the high- and medium- differentiated colorectal cancer tissues, Bcl-2 expression rate in B7-H1 negative group was higher than the positive group with significant difference (P<0.01). CONCLUSIONS: In colorectal carcinoma, Bcl-2, B7-H1, EGFR and VEGF were all expressed, independent from age and depth of invasion. However, the expression level of Bcl-2 and B7-H1 correlated with tissue differentiation, and the latter also had correlation with tumor staging. Meanwhile, the short-term follow-up showed that high expression of Bcl-2/B7-H1 existed in death cases. Therefore, the expression detection of Bcl-2, B7-H1 might provide a clear understanding of the biological behavior of colorectal cancer, and was important for the diagnosis, treatment and prognosis judgment of colorectal cancer.

Identification of Non-HIV Immunogens That Bind to Germline b12 Predecessors and Prime for Elicitation of Cross-clade Neutralizing HIV-1 Antibodies.

A fundamental challenge for developing an effective and safe HIV-1 vaccine is to identify vaccine immunogens that can initiate and maintain immune responses leading to elicitation of broadly neutralizing HIV-1 antibodies (bnAbs) through complex maturation pathways. We have previously found that HIV-1 envelope glycoproteins (Env) lack measurable binding to putative germline predecessors of known bnAbs and proposed to search for non-HIV immunogens that could initiate their somatic maturation. Using bnAb b12 as a model bnAb and yeast display technology, we isolated five (poly)peptides from plant leaves, insects, E. coli strains, and sea water microbes that bind to b12 putative germline and intermediate antibodies. Rabbit immunization with the (poly)peptides alone induced high titers of cross-reactive antibodies that neutralized HIV-1 isolates SF162 and JRFL. Priming rabbits with the (poly)peptides followed by boosts with trimeric gp140SF162 and then resurfaced Env (RSC3) induced antibodies that competed with mature b12 and neutralized tier 1 and 2 viruses from clade B, C and E, while control rabbits without (poly)peptide priming induced antibodies that did not compete with mature b12 and neutralized fewer isolates. The degree of competition with mature b12 for binding to gp140SF162 correlated with the neutralizing activity of the rabbit IgG. Reversing the order of the two boosting immunogens significantly affected the binding profile and neutralization potency of the rabbit IgG. Our study is the first to provide evidence that appears to support the concept that non-HIV immunogens may initiate immune responses leading to elicitation of cross-clade neutralizing antibodies.

Reconstitution and characterization of antibody repertoires of HIV-1-infected "elite neutralizers".

Around 3-5% HIV-1-infected individuals develop high titers of broadly neutralizing HIV-1 antibodies (bnAbs) during chronic infection. However, monoclonal antibodies (mAbs) isolated from such "elite neutralizers" do not, in most cases, depict the serum IgGs in neutralizing the virus. We hypothesize that HIV-1-specific antibodies in infected subjects may work in a population manner in containing the virus in vivo, and in vitro reconstituted antibody repertoires of "elite neutralizers" may mimic the sera in binding and neutralizing the virus. This study aims to investigate the antibody repertoires of three such "elite neutralizers" by reconstituting the immune antibody repertories in vitro followed by a comparative study of the recombinant library IgGs with the corresponding serum IgGs. We found that the recombinant library IgGs were much weaker than the serum IgGs in binding to envelope glycoproteins (Envs) and in neutralizing the virus and inhibiting Env-mediated cell-cell fusion. However, the sorted libraries composing of HIV-1-specific neutralizing antibodies (nAbs) in the three recombinant libraries exhibited comparable binding and inhibitory activities, as well as antibody-dependent cell-mediated cytotoxicity (ADCC), to the serum IgGs. The sorted library IgGs further showed neutralization profiles which are similar to those of the serum IgGs, but they were overall less potent than the serum IgGs. The sorted library IgGs and the serum IgGs bound weakly to the resurfaced Env gp120, RSC3, and did not bind to the CD4 binding site (CD4bs) knock-out mutant, ΔRSC3. Profiling with VRC01 binding site knock-out site mutants of gp120BaL indicates that, if there are any CD4bs bnAbs in these sera, they are more likely b12-like, but not VRC01-class bnAbs. Our results suggest that HIV-1-specific Ab-expressing B cells, especially potent nAb-expressing B cells may not be rich in the B cell repertoires of "elite neutralizers", but they may be highly active in producing nAbs in vivo. In vitro reconstituted HIV-1 nAb repertoires of "elite neutralizers" may be used in passive immunization to prevent HIV-1 infection. HIV-1 vaccine immunogens may be designed to target multiple neutralizing determinants to stimulate multiple B cell populations. HIV-1-specific antibodies induced by such immunogens may work in combination or synergistically in containing the virus.

Superior antitumor activity of a novel bispecific antibody cotargeting human epidermal growth factor receptor 2 and type I insulin-like growth factor receptor.

The humanized anti-HER2 monoclonal antibody (mAb) trastuzumab (Herceptin; Genentech) effectively inhibits human epidermal growth factor receptor 2 (HER2)-positive breast tumors. However, many patients responding to treatment often develop resistance. Cross-talk between type I insulin-like growth factor receptor (IGF-IR) and HER2 and elevated IGF-IR signaling have been implicated in tumor cell resistance to trastuzumab therapy. Previously, we reported that the anti-IGF-IR mAb m590 inhibits proliferation and migration of breast cancer MCF-7 cells in vitro. Here, we generated a "knobs-into-holes" bispecific antibody (Bi-Ab) against HER2 and IGF-IR by engineering trastuzumab and m590. We compared the effects of Bi-Ab treatment in vitro and in SKOV-3 HER2- and IGF-IR-overexpressing cancer xenograft mouse model with those of m590 and trastuzumab treatment alone or in combination. Bi-Ab effectively inhibited proliferation of HER2- and IGF-IR-overexpressing ovarian cancer SKOV-3 cells in vitro by ablating receptor phosphorylation and downstream PI3K/Akt and mitogen-activated protein kinase signaling. Bi-Ab more effectively inhibited cancer growth in SKOV-3 HER2- and IGF-IR-overexpressing cancer xenograft mouse model than m590 and trastuzumab alone or in combination. Mice bearing SKOV-3 HER2- and IGF-IR-overexpressing xenografts showed extensive and sustainable tumor regression when treated with Bi-Ab. Our results suggest that Bi-Ab has superior antitumor activity compared with monospecific antibodies, and cotargeting HER2 and IGF-IR may be clinically beneficial in minimizing the acquired resistance to trastuzumab therapy.

The potential of the human immune system to develop broadly neutralizing HIV-1 antibodies: implications for vaccine development.

OBJECTIVES AND DESIGN: Developing an effective HIV-1 vaccine that elicits broadly neutralizing HIV-1 human antibodies (bnAbs) remains a challenging goal. Extensive studies on HIV-1 have revealed various strategies employed by the virus to escape host immune surveillance. Here, we investigated the human antibody gene repertoires of uninfected and HIV-1-infected individuals at genomic DNA (gDNA) and cDNA levels by deep sequencing followed by high-throughput sequence analysis to determine the frequencies of putative germline antibody genes of known HIV-1 monoclonal bnAbs (bnmAbs). METHODS: Combinatorial gDNA and cDNA antibody libraries were constructed using the gDNAs and mRNAs isolated from uninfected and HIV-1-infected human peripheral blood mononuclear cells (PBMCs). All libraries were deep sequenced and sequences analysed using IMGT/HighV-QUEST software (http://imgt.org/HighV-QUEST/index.action). The frequencies of putative germline antibodies of known bnmAbs in the gDNA and cDNA libraries were determined. RESULTS AND CONCLUSION: The human gDNA antibody libraries were more diverse in heavy and light chain V-gene lineage usage than the cDNA libraries, indicating that the human gDNA antibody gene repertoires may have more potential than the cDNA repertoires to develop HIV-1 bnAbs. The frequencies of the heavy and kappa and lambda light chain variable regions with identical V(D)J recombinations to known HIV-1 bnmAbs were extremely low in human antibody gene repertoires. However, we found relatively high frequencies of the heavy and kappa and lambda light chain variable regions that used the same V-genes and had the same CDR3 lengths as known HIV-1 bnmAbs regardless of (D)J-gene usage. B-cells bearing B-cell receptors of such heavy and kappa and lambda light chain variable regions may be stimulated to induce HIV-1 bnAbs.

HIV-1 envelope glycoprotein variable loops are indispensable for envelope structural integrity and virus entry.

HIV-1 envelope (Env) glycoprotein is a trimer of heterodimer of gp120 and gp41, and derives from a trimeric glycoprotein precursor, gp160. Gp120 contains five conserved regions that are interspersed with 5 variable loop regions (V1-V5). Env variations in variable loop length and amino acid composition may associate with virus pathogenesis, virus sensitivity to neutralizing antibodies (nAbs) and disease progression. To investigate the role of each variable loop in Env function, we generated a panel of JRFL gp160 loop deletion mutants and examined the effects of each loop deletion on Env expression, Env cell surface display and Env-mediated virus entry into permissive cells. We found that deletion of V1 and V2 (ΔV1V2), or loop D (ΔlpD) abolished virus entry, the same effect as deletion of V3 (ΔV3), while deletion of V3 crown (ΔV3C) significantly enhanced virus assembly and entry. We further found that deletion of V4 (ΔV4) or V5 (ΔV5), or replacement of V4 or V5 with flexible linkers of the same lengths knocked out the receptor and coreceptor binding sites in gp120, but significantly enhanced the exposure of the N-trimer structure and the membrane proximal external region (MPER) in gp41. Although deletion of V4 or V5 did not affect Env expression, they negatively affected Env cell surface display, leading to the failure in virus assembly and subsequent entry. Taken together, we found that Env variable loops were indispensable for Env structural integrity and virus entry. Our findings may have implications for development of HIV-1 vaccine immunogens and therapeutics.

Identification of dominant antibody-dependent cell-mediated cytotoxicity epitopes on the hemagglutinin antigen of pandemic H1N1 influenza virus.

Antibody-dependent cell-mediated cytotoxicity (ADCC) bridges innate and adaptive immunity, and it involves both humoral and cellular immune responses. ADCC has been found to be a main route of immune protection against viral infections in vivo. Hemagglutinin (HA) of influenza virus is highly immunogenic and considered the most important target for immune protection. Several potent cross-reactive HA-specific neutralizing monoclonal antibodies (MAbs) have been reported, and their conserved neutralizing epitopes have been revealed, but there has been no report so far about ADCC epitopes on HA. Here we identified two dominant ADCC epitopes, designated E1 (amino acids [aa] 92 to 117) and E2 (aa 124 to 159), on HA of pandemic H1N1 influenza virus by epitope mapping of convalescent-phase plasma IgG antibodies from six H1N1-infected human subjects in China that exhibited different levels of ADCC activity. The E1 and E2 ADCC epitopes overlapped with immunodominant epitopes of HA. Depletion of purified patient plasma IgG antibodies with EBY100 yeast cells expressing E1 or E2 decreased the ADCC activity of the IgG antibodies. E1 and E2 sequences were found to be highly conserved in H1N1 strains but less so in other subtypes of influenza A viruses. Our study may aid in designing immunogens that can elicit antibodies with high ADCC activity. Vaccine immunogens designed to include the structural determinants of potent broadly neutralizing antibodies and ADCC epitopes may confer comprehensive immune protection against influenza virus infection.

Two-step self-assembly of iron oxide into three-dimensional hollow magnetic porous microspheres and their toxic ion adsorption mechanism.

Hollow magnetic porous Fe(3)O(4)/α-FeOOH microspheres, with abundant surface hydroxyl groups and carbonate-like species, were prepared using a simple template free solution method. The obtained magnetic microspheres were characterized by field emission scanning electron microscopy, transmission electron microscopy, nitrogen adsorption-desorption isotherms, vibrating sample magnetometry and X-ray diffraction. A two-step self-assembly mechanism for the microspheres was proposed based on the morphology of the products produced with different reaction times and the X-ray diffraction of the raw product grown at the initial stage. The toxic ion adsorption properties of the microspheres were investigated for As(V), Cr(VI), Cd(II) and Hg(II) ion removal. The adsorption mechanism was studied by an X-ray photoelectron spectrometer and Fourier transform infrared absorption spectroscopy . The results suggest that both the surface hydroxyl groups and the carbonate-like species participated in the ion-exchange process.