鼻咽癌放射治疗后颞叶损伤的质子磁共振波谱表现

目的初步报道鼻咽癌放射治疗后颞叶损伤的1HMRS表现,以期对临床诊断有所帮助,并初步探讨其临床意义.方法对13例鼻咽癌放射治疗后颞叶损伤病例行MRS检查.所有病例均在复旦大学附属华山医院检查,使用GE Singa Horizont 1.5 T超导型磁共振成像机,采用头颅正交线圈.所有病例均先进行MRI检查,再进行1HMRS检查,1HMRS采用点分辨波谱分析法及化学位移选择饱和水抑制法采集信号.成像参数为TR2000 ms,TE144 ms;成像时间为348 s;体素(voxel)大小2 cm×2 cm×2 cm;取样时将体素置于病灶的实质部分.并行对侧相应颞叶脑组织HMRS作对照(双侧颞叶损伤除外).结果首程放射距1HMRS检查的中位时间为42个月(32～172个月).13例中双侧颞叶病灶5例,共发现18个病灶.5例双侧颞叶损伤未进行正常脑组织14HMRS的对照检查,1例双侧颞叶损伤仅进行了1侧病灶检查,因此13例共进行了17个病灶区和8个正常脑组织HMRS检查.HMRS表现为病灶侧NAA/Cr均值为1.33±0.28,而对照脑组织的NAA/Cr均值为1.78±0.34,二者差异有显著性意义(t=3.22,P=0.005).3个病灶出现NAA、Cho、Cr峰消失(其中2个病灶伴Lip峰).8例有对照的颞叶损伤中6例病灶侧Cho/Cr比值低于对照侧.4个病灶出现Lip峰,4个病灶出现倒置的Lac峰,4个病灶出现MI峰.结论鼻咽癌放射治疗后颞叶损伤均有HMRS异常,主要表现为NAA/Cr的下降,大部分病例伴Cho/Cr下降,可出现Lip峰、倒置的Lac峰及MI峰.HMRS可能在放射性颞叶损伤诊断中有一定价值.     

Biomolecular modification of p(AAm-co-EG/AA) IPNs supports osteoblast adhesion and phenotypic expression

Interpenetrating polymer networks (IPNs) were designed to resist materials fouling caused by non-specific protein adsorption, and indiscriminate cell or bacterial adhesion. These IPNs were thin adherent films ( ~ 20 nm) comprised of acrylamide (AAm), ethylene glycol (EG), and acrylic acid (AA) grafted to either silicon waters or quartz substrates via photoinitiated free radical polymerization. These networks were further modified to promote specific cell adhesion by tethering bioactive groups such as peptides that mimic cell-binding domains found on extracellular matrix molecules. As a specific example of biomolecular surface engineering, peptides from the cell-binding domain of bone sialoprotein were tethered to a p(AAm-co-EG/AA) IPN to control cell behavior at the surface. The networks were characterized by contact angle measurements, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy to convey information on IPN wettability, thickness, and chemistry. The surface characterization data supported the theory that the PEG/AA layer formed an IPN with the underlying p(AAm) network, and after graft modification of this IPN with diamino PEG (PEG(NH₂)₂), the PEG(NH₂)₂ chains were enriched at the surface. Rat calvarial osteoblasts attached to Arg-Gly-Asp (RGD) modified IPNs at levels significantly greater than on clean quartz, Arg-Gly-Glu (RGE) modified, or the PEG(NH₂)₂ modified IPN, with or without serum in the media. Cells maintained in media containing 15% fetal bovine serum (FBS) proliferated, exhibited nodule formation, and generated sheets of mineralized extracellular matrix (ECM) with the addition on β-glycerophosphate to the media. Cell adhesion and mineralized ECM formation were specifically dependent on the peptide sequence present at the surface.     

海绵共附生真菌Penicillium chrysogenum次级代谢产物的化学成分及生物活性研究

目的 对分离自西沙隋氏蒂壳海绵共附生真菌Penicilliumchrysogenum的次级代谢产物进行化学成分及其生物活性研究,以期发现结构特异并且活性良好的次级代谢产物。方法 对隋氏蒂壳海绵共附生真菌Penicilliumchrysogenum用真菌2号培养基发酵,发酵后的菌丝体采用溶剂提取、萃取和现代色谱分离纯化手段,再运用现代核磁波谱技术并结合高分辨质谱鉴定化合物结构。基于微阵列技术的表面等离子体共振成像（SPRi）系统,检测化合物与肿瘤相关蛋白的相互作用,并提供化合物与肿瘤相关蛋白的结合动力学数据。结果 通过分离隋氏蒂壳海绵共附生真Penicilliumchrysogenum的菌丝体提取物,从中分离鉴定了7个单体化合物,鉴定结果为conidiogenone（1）、2-acetylquinazolin-4(3H)-one（2）、15β-hydroxyl-(22E,24R)-ergosta-3,5,8,22-tetraen-on（3）、ergosta-4,6,8(14),22-tetraen-3-one（4）、 2-((2E,4E)-hexa-2,4-dienoyl)-5,6-dihydroxy-4,6-dimethylcyclohex-4-ene-1,3-dione（5）、(22E)-5α,8α-epidioxyergosta-6,22-dien-3β-ol（6）、2-(1-hydroxyethyl)quinazolin-4(3H)-one（7）。结论 化合物3和6是从Penicillium属内第一次分离得到,化合物4是从真菌Penicilliumchrysogenum中第一次分离得到,化合物5与肿瘤蛋白VEGFR-1、FGFR有亲和作用,KD的数值分别为7.78×10-3和1.44×10-1 μmol/L。     

Stress granule formation,disassembly, and composition are regulated by alphavirus ADP-ribosylhydrolase activity

While biomolecular condensates have emerged as an important biological phenomenon, mechanisms regulating their composition and the ways that viruses hijack these mechanisms remain unclear. The mosquito-borne alphaviruses cause a range of diseases from rashes and arthritis to encephalitis, and no licensed drugs are available for treatment or vaccines for prevention. The alphavirus virulence factor nonstructural protein 3 (nsP3) suppresses the formation of stress granules (SGs)—a class of cytoplasmic condensates enriched with translation initiation factors and formed during the early stage of infection. nsP3 has a conserved N-terminal macrodomain that hydrolyzes ADP-ribose from ADP-ribosylated proteins and a C-terminal hypervariable domain that binds the essential SG component G3BP1. Here, we show that macrodomain hydrolase activity reduces the ADP-ribosylation of G3BP1, disassembles virus-induced SGs, and suppresses SG formation. Expression of nsP3 results in the formation of a distinct class of condensates that lack translation initiation factors but contain G3BP1 and other SG-associated RNA-binding proteins. Expression of ADP-ribosylhydrolase–deficient nsP3 results in condensates that retain translation initiation factors as well as RNA-binding proteins, similar to SGs. Therefore, our data reveal that ADP-ribosylation controls the composition of biomolecular condensates, specifically the localization of translation initiation factors, during alphavirus infection.

Biomolecular condensates are prevalent in cells and critical for a range of cellular functions, including RNA metabolism, embryonic cell fate specification, and neuronal activity (1–3). While condensates often dynamically exchange components with the surrounding milieu, the overall composition of these cellular structures remains distinct (4). How cells control the specific composition of these condensates remains unclear. Stress granules (SGs), one of the best characterized biomolecular condensates, are RNA–protein assemblies formed in response to a variety of environmental cues (1). While SG composition can vary with the type of stress cue (5), certain common components, such as Ras GTP-activating protein-binding proteins G3BP1/2, are essential for formation of SGs (6, 7). Dysregulation of SG formation and disassembly is implicated in the pathogenesis of diseases, including viral infection, cancer, and neurodegeneration (2, 8–10).SG formation and disassembly are tightly regulated during viral infection, often reflecting cellular translation status (11–14). In the early phase of many viral infections, the presence of double-stranded viral RNAs (vRNAs) activate protein kinase R (PKR), resulting in eIF2α phosphorylation, messenger RNA (mRNA) translation inhibition, and formation of SGs enriched with translation initiation factors such as eIF3b. However, in later infection stages, many viruses instead suppress SG formation or disassemble SGs altogether. The mechanisms underlying this switch, and its physiological function, remain unclear.SG formation and disassembly are regulated by posttranslational modifications of proteins, including those that conjugate simple chemical groups, attach polypeptides, and add nucleotides as in the case of ADP-ribosylation (15–21). ADP-ribosylation refers to the addition of one or more ADP-ribose units onto proteins (22–24). In humans, ADP-ribosylation is accomplished primarily by a family of 17 ADP-ribosyltransferases, commonly known as poly(ADP-ribose) polymerases (PARPs). SG components are specifically ADP-ribosylated, and ADP-ribose polymers [i.e., poly(ADP-ribose) or PAR], five PARPs and two isoforms of the degradative enzyme PAR glycohydrolase (PARG) have been localized to these condensates (17, 25–27). Overexpression of these PARPs and PARG isoforms induces and suppresses SG formation, respectively, while PARG knockdown delays SG disassembly (17, 26). The noncovalent interaction between PAR and proteins facilitates SG targeting (25–27). For example, PAR-mediated targeting regulates TDP-43 localization to SGs and prevents the formation of pathological aggregates in amyotrophic lateral sclerosis (26, 27).The mosquito-borne alphaviruses, which cause a range of diseases from rashes and arthritis to encephalitis, induce SG formation early in infection and later initiate SG disassembly (11, 14, 28, 29). Previous studies have identified the alphaviral nonstructural protein 3 (nsP3), a key factor for virus replication and virulence (30–32), as able to suppress SG formation (28, 33–35). The alphaviral nsP3 is a tripartite protein composed of a highly conserved macrodomain (MD) in the N terminus, a central zinc-binding domain (ZBD), and a C-terminal hypervariable domain (HVD; ref. 30). Recent studies indicate that the HVD, which is of low complexity, directs alphaviral nsP3 binding to host SG proteins (30, 36). For example, the HVD of chikungunya virus (CHIKV) binds the essential SG components G3BP1 and G3BP2 (33, 37). Given that nsP3 expression increases over the course of viral infection, it has been proposed that nsP3 sequesters G3BP1/2, resulting in the suppression of SG formation during the late phase of infection (28, 29, 34).Here, we report that the expression of the G3BP-binding HVD alone does not suppress SG formation; rather, expression of the N-terminal MD alone can trigger the suppression of this biomolecular condensate. The structural integrity of SGs is dependent on ADP-ribosylation (17), and we and others recently found that the viral MD can remove single ADP-ribose groups, and possibly PAR, from ADP-ribosylated proteins (31, 38–40). We therefore hypothesized that MD ADP-ribosylhydrolase activity is required to suppress SG formation across stress conditions, with G3BP1 being a key target substrate. Indeed, we find that MD ADP-ribosylhydrolase activity is critical for disassembling SGs formed by G3BP1 expression and during viral infection. Consistent with this premise, live cell imaging revealed that SGs persist in cells infected with a hydrolase-deficient recombinant CHIKV. ADP-ribosylhydrolase activity is required for altering the composition of biomolecular condensates in nsP3-expressing or virus-infected cells and specifically regulates translation factor localization. Together, these data argue that nsP3 ADP-ribosylhydrolase activity modulates SG formation, disassembly, and composition.     

Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies against Delta and Omicron in Mice

As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We previously developed highly thermo-tolerant monomeric and trimeric receptor-binding domain derivatives that can withstand 100 °C for 90 min and 37 °C for four weeks and help eliminate cold-chain requirements. We show that mice immunised with these vaccine formulations elicit high titres of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an average 14.4-fold reduction in neutralisation against BA.1.1 for the three monomeric antigen-adjuvant combinations and a 16.5-fold reduction for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for upcoming Phase I human clinical trials and that there is potential for increasing the efficacy with vaccine matching to improve the responses against emerging variants. These findings are consistent with in silico modelling and AlphaFold predictions, which show that, while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible and also carries the risk of eliciting unwanted antibodies against the oligomerisation domain.     

HBV相关糖基转移酶Glt25D2的核苷酸单糖结合活性分析

目的体外克隆表达人类糖基转移酶Glt25D2,利用Biacore分析系统对其活化单糖及钙离子结合活性进行分析。方法构建Glt25D2原核表达载体pET32a-Glt25D2,转化大肠埃希菌BL21,克隆表达糖基转移酶Glt25D2。离心集菌,制备蛋白样品,进行SDS-PAGE电泳分析;融合蛋白线性梯度洗脱,Ni-NTA柱纯化,后做Western blot鉴定。用纯化后的Glt25D2融合蛋白包被CM5芯片,分别用不同浓度的活性单糖及钙离子灌注芯片,利用Biacore生物分子相互作用分析仪分析Glt25D2的活化单糖及钙离子结合活性。结果体外成功表达人类糖基转移酶Glt25D2融合蛋白。Biacore分析显示,该糖基转移酶与400、200、100、50μg/ml唾液酸的结合活性分别为108、71、50和20 RU,与200、100、50、0μg/ml钙离子的结合活性分别为37、20、10和0 RU。结论人类糖基转移酶Glt25D2重组蛋白具有较强的钙离子及唾液酸结合活性。     

Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins

Single-molecule (SM) microscopy allows outstanding insight into biomolecular mechanisms in cells. However, selective detection of single biomolecules in their native environment remains particularly challenging. Here, we introduce an easy methodology that combines specific targeting and nanometer accuracy imaging of individual biomolecules in living cells. In this method, named complementation-activated light microscopy (CALM), proteins are fused to dark split-fluorescent proteins (split-FPs), which are activated into bright FPs by complementation with synthetic peptides. Using CALM, the diffusion dynamics of a controlled subset of extracellular and intracellular proteins are imaged with nanometer precision, and SM tracking can additionally be performed with fluorophores and quantum dots. In cells, site-specific labeling of these probes is verified by coincidence SM detection with the complemented split-FP fusion proteins or intramolecular single-pair Förster resonance energy transfer. CALM is simple and combines advantages from genetically encoded and synthetic fluorescent probes to allow high-accuracy imaging of single biomolecules in living cells, independently of their expression level and at very high probe concentrations.     

~1H磁共振波谱在胶质瘤放疗后复发和放射性脑坏死鉴别中的初步应用

目的　探讨质子磁共振波谱 (1HMRS)对胶质瘤放疗后复发和放射性脑坏死鉴别诊断的价值。方法　 15例有脑部放疗史 ,临床及CT、MRI难以判断为肿瘤复发或放射性脑坏死的患者 ,5例病史明确的放射性脑坏死的患者 ,均行1HMRS检查。结果　前 15例经手术证实 ,14例为胶质瘤 ,1例放射性脑坏死 ,1HMRS诊断正确。 (1) 14例胶质瘤在1HMRS上均表现为明显增高的胆碱(Cho)峰 ,氮乙酰门冬氨酸 (NAA)、肌酸 (Cr)峰下降或消失 ,Cho/Cr比值升高 ,NAA/Cr比值降低 ;12例出现乳酸 (Lac)峰。 (2 )放射性脑坏死表现为 :5例Cho、NAA、Cr下降或消失 ,出现脂质 (Lipid)峰 ;1例Cho、NAA、Cr峰均消失 ,仅表现一较平坦的曲线 ,无Lac峰。结论　1HMRS对胶质瘤放疗后复发和放射性脑坏死的鉴别有重要价值。