Dromedary camel nanobodies broadly neutralize SARS-CoV-2 variants

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is a trimer of S1/S2 heterodimers with three receptor-binding domains (RBDs) at the S1 subunit for human angiotensin-converting enzyme 2 (hACE2). Due to their small size, nanobodies can recognize protein cavities that are not accessible to conventional antibodies. To isolate high-affinity nanobodies, large libraries with great diversity are highly desirable. Dromedary camels (Camelus dromedarius) are natural reservoirs of coronaviruses like Middle East respiratory syndrome CoV (MERS-CoV) that are transmitted to humans. Here, we built large dromedary camel V_HH phage libraries to isolate nanobodies that broadly neutralize SARS-CoV-2 variants. We isolated two V_HH nanobodies, NCI-CoV-7A3 (7A3) and NCI-CoV-8A2 (8A2), which have a high affinity for the RBD via targeting nonoverlapping epitopes and show broad neutralization activity against SARS-CoV-2 and its emerging variants of concern. Cryoelectron microscopy (cryo-EM) complex structures revealed that 8A2 binds the RBD in its up mode with a long CDR3 loop directly involved in the ACE2 binding residues and that 7A3 targets a deeply buried region that uniquely extends from the S1 subunit to the apex of the S2 subunit regardless of the conformational state of the RBD. At a dose of ≥5 mg/kg, 7A3 efficiently protected transgenic mice expressing hACE2 from the lethal challenge of variants B.1.351 or B.1.617.2, suggesting its therapeutic use against COVID-19 variants. The dromedary camel V_HH phage libraries could be helpful as a unique platform ready for quickly isolating potent nanobodies against future emerging viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of COVID-19 (1, 2) that enters human cells by binding its envelope anchored type I fusion protein (spike) to angiotensin-converting enzyme 2 (ACE2) (3, 4). The SARS-CoV-2 spike is a trimer of S1/S2 heterodimers with three ACE2 receptor-binding domains (RBDs) attached to the distal end of the spike via a hinge region that allows conformational flexibility (4). In the all-down conformation, the RBDs are packed with their long axes contained in a plane perpendicular to the axis of symmetry of the trimer. Transition to the roughly perpendicular up conformation exposes the receptor-binding motif (RBM), located at the distal end of the RBD, which is sterically occluded in the down state. Numerous neutralizing antibodies targeting the spike, particularly its RBD, have been developed to treat COVID-19 using common strategies such as single B cell cloning, animal immunization, and phage display (5–9). Most vaccines, including those that are messenger RNA based, are designed to induce immunity against the spike or RBD (10–12). However, emerging SARS-CoV-2 variants such as D614G, B.1.1.7 (Alpha, United Kingdom), B.1.351 (Beta, South Africa), and P.1 (Gamma, Brazil) have exhibited increased resistance to neutralization by monoclonal antibodies or postvaccination sera elicited by the COVID-19 vaccines (13, 14). Monoclonal antibodies with Emergency Use Authorization for COVID-19 treatment partially (Casirivimab) or completely (Bamlanivimab) failed to inhibit the B.1.351 and P.1 variants. Similarly, these variants were less effectively inhibited by convalescent plasma and sera from individuals vaccinated with a COVID-19 vaccine (BNT162b2) (13). The B.1.617.2 (Delta, India) variant became the prevailing strain in many countries (15). Highly effective and broadly neutralizing antibody therapy is urgently demanded for COVID-19 patients.Due to their small size and unique conformations, camelid V_HH single-domain antibodies (also known as nanobodies) can recognize protein cavities that are not accessible to conventional antibodies (16). To isolate high-affinity nanobodies without a need for further affinity maturation, it is highly desirable to construct large nanobody libraries with great diversity. Dromedary camels have been found as potential natural reservoirs of Middle East respiratory syndrome CoV (MERS-CoV) (17). We speculated that dromedary camels would be an ideal source of neutralizing nanobodies against coronaviruses. In the present study, we built large camel V_HH single-domain antibody phage libraries with a diversity of over 10¹¹ from six dromedary camels (Camelus dromedarius), three males and three females, with ages ranging from 3 mo to 20 y. We used both the SARS-CoV-2 RBD and the stabilized spike ectodomain trimer protein as baits to conduct phage panning for nanobody screening. Among all the binders, we found NCI-CoV-7A3 (7A3), NCI-CoV-1B5 (1B5), NCI-CoV-8A2 (8A2), and NCI-CoV-2F7 (2F7) to be potent ACE2 blockers. In addition, these dromedary camel nanobodies displayed potent neutralization activity against the B.1.351 and B.1.1.7 variants and the original strain (Wuhan-Hu-1). The cryoelectron microscopy (cryo-EM) structure of the spike trimer protein complex with these V_HH nanobodies revealed two distinct nonoverlapping epitopes for neutralizing SARS-CoV-2. In particular, 7A3 recognizes a unique and deeply buried region that extends to the apex of the S2 subunit of the spike. Combined treatment with 7A3 and 8A2 shows more potent protection against various variants in culture and mice infected with the B.1.351 variant. Interestingly, 7A3 alone retains its neutralization activity against the lethal challenge of the B.1.617.2 variant in mice.     

2种不同弹力纤维染色方法对肿瘤侵犯判读结果的对比研究

目的 比较Victoria blue法(A组)和Verhoeff法(B组)弹力纤维染色在染色过程、染色时间、染色效果方面对肺癌胸膜侵犯和结直肠癌浆膜或静脉侵犯判读的差异。方法 A组、B组均分别纳入肺癌怀疑胸膜侵犯标本50例和结直肠癌怀疑浆膜或静脉侵犯标本20例,进行弹力纤维染色。对比2组染色过程、染色时间、染色效果;根据染色稳定性、清晰度、对比度等综合评价胸膜侵犯和浆膜或静脉侵犯情况,并对染色质量及判读准确性进行评分。结果 A组比B组操作简单,染色质量稳定,肿瘤细胞与脏层胸膜、浆膜或静脉的关系显示更清晰。A组判读评分高于B组,差异有统计学意义(P<0.05)。结论 Victoria blue法染色有助于准确评估肺癌的胸膜侵犯和结直肠癌浆膜或静脉的侵犯,提高判读准确性。     

Arthroscopic Modified Double‐Row Biceps Tenodesis versus Labral Repair for the Treatment of Isolated Type II SLAP Lesions in Non‐Overhead Athletes

ObjectiveTo evaluate the postoperative efficacy and the clinical outcomes of arthroscopic modified double‐row biceps tenodesis versus labral repair.MethodsA retrospective study was conducted in 56 patients with isolated type II superior labrum anterior and posterior (SLAP) lesions from March 2015 to November 2018. Thirty patients (male:female = 17:13) were treated with labral repair, and 26 patients (male:female = 15:11) were treated with modified double‐row biceps tenodesis. The average age of the labral repair group and the modified double‐row biceps tenodesis group were 42.8 ± 10.6 and 40.9 ± 10.2 years, respectively. Pre‐ and postoperative assessments with the visual analog scale (VAS), University of California Los Angeles (UCLA), and American Shoulder and Elbow Surgeons (ASES) scores were compared between the two treatment groups. Additional outcome measures included patient satisfaction, the time to return to previous activities, workers'' compensation status, and postoperative complications.ResultsAt a 2‐year follow‐up, the tenodesis group showed significant differences in postoperative VAS (1.5 to 1.8, respectively; p = 0.008), patient satisfaction (92.3% vs. 46.7%, p < 0.001), and recovery time to return to their previous activities (6.8 ± 1.8 vs. 8.1 ± 1.5, p = 0.007) compared to the labral repair group; however, there was no significant difference in postoperative ASES and UCLA scores between the two groups. Additionally, one patient in the tenodesis group developed persistent postoperative stiffness, which was resolved by conservative treatment. In the labral repair group, two patients presented with persistent postoperative night pain, three developed persistent postoperative stiffness, and two required a subsequent capsular release.ConclusionsCompared with the labral repair group, the arthroscopic modified double‐row biceps tenodesis showed more encouraging postoperative pain reduction, earlier recovery to previous activities, and higher patient satisfaction.     

技术行政综合干预对不合理医嘱干预成功率的影响调查

目的调查在技术措施基础上,联用行政措施干预静脉药物调配中心(PIVAS)不合理医嘱的效果。方法将2012年8月至11月医院老年病区药房静脉药物调配中心61 962条医嘱分为对照组和试验组,分别采用技术措施和技术行政综合措施对不合理医嘱进行干预,并统计分析。结果采用技术行政综合措施干预后,试验组不合理医嘱比例为1.27%,比对照组下降1.28%;不合理医嘱干预成功率为16.43%,比对照组提高6.65%,差异有统计学意义(P<0.05)。结论综合干预措施,对于提高不合理医嘱干预的成功率具有一定的促进作用。     

Abnormal neurogenesis in the dentate gyrus of adult mice lacking 1,25-dihydroxy vitamin D3 (1,25-(OH)2 D3)

In this study, we employed 1α-hydroxylase knockout (1α-(OH)ase(-/-) ) mice to investigate the influence of 1,25-dihydroxy vitamin D(3) (1,25-(OH)(2) D(3) ) deficiency on the adult neurogenesis in the hippocampal dentate gyrus (DG). The numbers of both 24-hr-old BrdU(+) cells and proliferating cell nuclear antigen positive cells in 8-week-old 1α-(OH)ase(-/-) mice increased approximately twofold compared with wild-type littermates. In contrast, the numbers of 7- and 28-day-old BrdU(+) cells in 1α-(OH)ase(-/-) mice decreased by 50% compared with wild-type mice, while the proportion of BrdU(+) /NeuN(+) cells in BrdU(+) population showed no difference between 1α-(OH)ase(-/-) and wild-type mice. Apoptotic cells in the subgranular zone (SGZ) of DG markedly increased in 1α-(OH)ase(-/-) mice. Replenishment of 1,25-(OH)(2) D(3) , but not correction of serum calcium and phosphorus levels, completely prevented changes in the neurogenesis in 1α-(OH)ase(-/-) mice. The absence of 1,25-(OH)(2) D(3) led to an increase in the expression of L-type voltage-gated calcium channel (L-VGCC) and a decrease in the nerve growth factor (NGF) mRNA level. Treatment with the L-VGCC inhibitor nifedipine blocked the increased cell proliferations by 1,25-(OH)(2) D(3) deficiency. Administration of NGF significantly attenuated the loss of newborn neurons in 1α-(OH)ase(-/-) mice.     

从4340份精液检查结果分析探讨不育的主要因素

 目的 回顾性分析4340份男性生殖系统疾病患者精液标本实验室检查结果,探讨不育的主要因素。方法 采用西班牙SCA精液分析系统,按照第5版《WHO人类精液检查与处理实验手册》要求对收集的4340份精液标本进行常规分析,用迈瑞BS-480生化分析仪检测精浆果糖含量。结果 4340份就诊患者标本,全完正常的1857份(43％),异常的2483份(57％),异常标本中,液化异常44份(1.77％),pH异常54份(2.17％),精液量异常303份(12.20％),密度异常616份(24.81％),活力异常1423份(57.31％),精浆果糖异常411份(16.55％),无精子症138份(5.56％),血精52份(2.09％),白细胞精子症89份(3.58％)。结论 精液质量降低是导致不育的主要因素,其中以精子活力影响最大。     

脑脊液细胞学在蛛网膜下腔出血中的应用价值

目的：应用细胞玻片离心仪和50例蛛网膜下腔出血（SAH）患者进行了不同时期脑脊液细胞学检查。方法：常规抽取脑脊液,经FMU-5细胞玻片离心仪直接离心制片,进行MGG染色,进行细胞分类。结果：蛛网膜下腔出血后脑脊液中白细胞在不同时期的细胞特征是：急性期：大量红细胞及高比例的嗜中性粒细胞期,嗜中性粒细胞比率在70%以上,48～72 h达高峰,一周后逐渐消失。吸收期：激活单核细胞增多,可出现红细胞吞噬细胞及特征性细胞-含铁血黄素吞噬细胞,持续2～3周。恢复期：主要以激活单核细胞为主,持续数周或几月。结论：应用该法进行脑脊液细胞学检查,可给临床提供一个直观、准确的诊断依据。对蛛网膜下腔出血病人的病理学监测、鉴别诊断和治疗具有重要价值。     

肺鳞癌小肠转移并消化道大出血1例

肺癌转移常见于脑、骨、肝、肾上腺等,而转移至小肠则非常罕见。我科2009—03收治1例肺鳞癌小肠转移并消化道大出血误诊原发小肠癌患者。