Study on Outer Membrane Protein Patterns of Escherichia coli O38,O53 and O75 Isolated from Chickens

[Objective] This study aimed to investigate the outer membrane protein （OMP） patterns of Escherichia coli 038, 053 and 075 isolates from chickens. [Method] Eight pathogenic E. coil isolates with various serotypes were used as experimental materials to extract OMP by using supersonic schizolysis method and Sarcosyl. After SDS-PAGE electrophoresis, OMP patterns of the extracted products were determined based on the OMP model diagram. [Result] OMP of eight E. coil isolates with three serotypes were divided into three patterns, to be specific, 2 075 isolates respectively belonged to OMP-I and OMP-II pattern, 1 053 isolate belonged to OMP-II pattern, and 5 038 isolates belonged to OMP-I and OMP-III pattern. [Conclusion] Experimental results showed that E. coli isolates with the same serotype may belong to completely different OMP patterns, while serologically unrelated isolates may belong to the same OMP pattern. OMP of E. coil isolates with the same serotype may generate genetic differentiation; in addition, OMP of E. coli isolates with different serotypes may have different genetic correlation.

Fabrication and applications of the protein patterns

Protein has been widely used for fabricating patterned structures since it is one of the most important macromolecules in living organisms,and protein patterns possess potential applications in many fields such as medical diagnosis,tissue engineering,biosensors,and medical screening.At present,there are two fashions to fabricate protein patterns:one is grafting the protein to the microstructure which is prepared by micro-fabrication techniques;the other one is achieving the patterned protein structures directly.Here we provide an overview on current status of the fabrication techniques and the applications of the protein patterns,and then give an outlook on the development of the fabrication techniques and the prospective applications of the protein patterns in future research.

SDS-PAGE PROTEIN PATTERN AND ITS ANTIGENICITY ANALYSIS OF DIFFERENT ISOLATES OF SCHISTOSOMA JAPONICUM IN CHINA

Homogenates prepared from S. japonicum adult worms ofdifferent isolates from Anhui, Hubei, Guangxi, Yunnan andSichuan Provinces were analyzed by SDS-PAGE and enzymelinked immunoelectrotransfer blot (EITB) tested with rabbitanti-snails antibody. The results of SDS-PAGE indicated thatwith silver staining both male and female worms of Guangxiisolate showed some definite differences in their protein profile,namely, absence of one band between 50-75 kDa in maleworms and marked reduction in quantity of > 110 and 30 kDabands in female worms. There was no obvious differenceamong other isolates both in male and female worms. TheEITB patterns were similar in S. japonicum of Anhui andHubei, and it was also the case with isolates from Yunnan andSichuau, except that Yuunan female worms had a distinct band

Pattern of expression of the CREG gene and CREG protein in the mouse embryo

Background The cellular repressor of ElA-stimulated genes(CREG) is a secreted glycoprotein that inhibits cell proliferation and/or enhances differentiation.CREG is widely expressed in adult tissues such as the brain,heart, lungs,liver,intestines and kidneys in mice.We investigated the level of CREG expression during mouse embryogenesis and its distribution at 18.5 days post coitus(dpc).Methods Immunohistochemical staining with diaminobenzidine,western blotting and reverse transcription-polymerase chain reaction were used.Results CREG expression was rst detected in mouse embryos at 4.5 dpc.It was expressed at almost all stages up to 18.5 dpc.The level of CREG was found to increase gradually and was highest at 18.5 dpc.Western blotting showed that the CREG protein was expressed at higher levels in the brain,heart,intestines and kidneys than in the lungs and liver at 18.5 dpc.In 9.5 dpc embryos,CREG was expressed only in the endothelial cells of blood vessels,after the vascular lumen had formed.With advanced differentiation, vascular smooth muscle cells developed in the embryonic vascular structures;the expression of smooth muscle a-actin protein and CREG were positive and increased gradually in 10.5 dpc embryonic vessels.CREG expression in the embryonic blood vessels peaked at 15.5 dpc and was reduced slightly at 18.5 dpc.Conclusions These results indicate that CREG is expressed during mouse embryogenesis and might participate in the differentiation of these organs during embryogenesis.

Comparative analysis of bed density, total phenol content and protein expression pattern in <i>Posidonia oceanica</i>(L.) Delile

Posidonia oceanicameadows are experiencing a progressive decline, and monitoring their status is crucial for the maintenance of theseecosystems. We performed a comparativeanalysis of bed density, total phenol content and protein expression pattern to assess the conservation status ofPosidoniaplants from the S. Marinella (Rome, Italy) meadow. The total phenol content was inversely related to maximum beddensity, confirming the relationship betweenhigh phenol content and stressful conditions. In addition, protein expression pattern profilesshowed that the number of differentially expressed proteins was dramatically reduced in the latest years compared to previous analyses. Our results support the usefulness of integrating solid descriptors, such as phenol content, with novel biochemical/molecular approaches in the monitoring of meadows.

Prediction Method of Protein Disulfide Bond Based on Pattern Selection

The effect of the different training samples is different for the classifier when pattern recognition system is established. The training samples were selected randomly in the past protein disulfide bond prediction methods, therefore the prediction accuracy of protein contact was reduced. In order to improve the influence of training samples, a prediction method of protein disulfide bond on the basis of pattern selection and Radical Basis Function neural network has been brought forward in this paper. The attributes related with protein disulfide bond are extracted and coded in the method and pattern selection is used to select training samples from coded samples in order to improve the precision of protein disulfide bond prediction. 200 proteins with disulfide bond structure from the PDB database are encoded according to the encoding approach and are taken as models of training samples. Then samples are taken on the pattern selection based on the nearest neighbor algorithm and corresponding prediction models are set by using RBF neural network. The simulation experiment result indicates that this method of pattern selection can improve the prediction accuracy of protein disulfide bond.

Effects of BMP-2 Patterns on Bovine Chondrocytes Adhesion and Alignment

Striped bone morphogenetic protein-2 （BMP-2） patterns are created on polystyrene （PS） surfaces by microcontact printing （μCP） to investigate the influences of the protein patterns on bovine chondrocytes behaviors. Due to the excellent ability of BMP-2 to recruit cells and the limited ability of blank PS areas to bind ceils, bovine chondrocytes preferentially attach on the protein areas, leading to formation of cell patterns and elongated cell morphologies to some degree. The BMP-2 protein stripe can guide bovine chondrocytes adhesion and alignment. The pattern dimensions can significantly affect the cell adhesion and spread. The protein stripe width mainly controls the cell elongation and orientation while the pattern spacing mainly affects the cell spread towards neighboring stripes. Therefore, the cell morphology and distribution direction can be controlled by precisely designing the pattern shapes and sizes. We believe that the present study could fred applications for surface modification of biomaterials＇ surfaces to create the bioactive patterns to control chondrocytes adhesion, spreading and even cell function. It may be helpful for the development of novel biomaterials for cartilage repair.

Applications of Pattern Recognition in Drug Discovery

This is a brief account of the plenary talk given to the meeting of the Chinese Society of Chemical Science and Technology held in Oxford on 6 October 2001. The talk covered the application of pattern recognition techniques to discover molecules which will bind to the binding sites of proteins. Three situations were considered: the structure of the protein being unknown; the structure known but the binding site unknown; and finally, and this is the most important case for the future, both the structure and nature of the target site available in atomic detail. For this case we have developed a massively distributed computer program using a screensaver which now involves over one million personal computers, including over a thousand in China. The project will involve the screening of 3 5 billion small molecules against 16 protein targets, all of which are implicated in the process of cancer.

Characterization and Expression Analysis of Four Glycine-Rich RNA-Binding Proteins Involved in Osmotic Response in Tobacco (Nicotiana tabacum cv. Xanthi)

Plants have developed many signals and specific genes＇ regulations at both transcriptional and post-transcriptional levels in order to tolerate and adapt to various environmental stresses. RNA-binding proteins （RBPs） play crucial roles in the post- transcriptional regulation via mRNA splicing, polyadenylation, sequence editing, transport, mRNA stability, mRNA localization, and translation. In this paper, four cDNAs of glycine-rich RNA-binding proteins （GR-RBPs）, named NtRGP-la, -lb, -2, and -3, were isolated from Nicotiana tabacum by RT-PCR analysis, and special emphases were given to the sequences alignment, phylogenetic analysis and gene expression. Sequences alignment revealed minor difference of cDNA sequences, but no difference of deduced proteins between N. sylvestris and N. tabacum. Phylogenetic alignment revealed that four cDNAs in tobacco were clustered into two different groups. NtRGP-2 and -3 were evolutionarily closest to Arabidopsis GR-RBPs genes and related to animal GR-RBPs genes, while NtRGP-la and -lb were closest to Gramineae GR-RBPs genes. The expression analyses of these four NtRGPs in response to different abiotic stresses revealed the similar expression pattern. Moreover, the four NtRGPs, especially NtRGP-la and NtRGP-3, were strongly induced by stresses including water, wound, cold, and high temperature, weakly induced by PEG, drought and SA, while reduced by NaC1 and unaffected by ABA treatment. The fact that all of these abiotic stresses included in our experiments affected the water balance and resulted in osmotic stress on cellular level, suggests that NtRGPs in tobacco should be a family of crucial osmosis-related proteins, and may play a key role in signal transduction with ABA-independent pathway under abiotic stresses.

Bacterial Surface Layer Proteins: A Promising Nano-Technological Tool for Bio-Sensing Applications

The phenomenal rise in the demand of biosensors accelerated their rapid development and immersive applications in the myriads of fields. The essential requirement of developing efficient bio-sensing platform is to find stable well organized interfacial architecture that can serve as an excellent matrix for binding and recognizing biomolecules. In this context, the enormous potential has been envisaged in surface layer proteins that represented themselves as most primitive and simplest self-assembled system with repetitive physicochemical properties for the molecular functionalization of surfaces and various interfaces. The prominence of S-layer proteins has been broadened by integrating genetic engineering approaches for the fine tuning of functional groups and protein domains in geometrically well-defined manner. The efficient and stable binding of various nanomaterials with S-layers in regular arrays has led to paradigmatic shift in their nano-biotechnological sensing applications. More recently, functional S-layer supported lipid membranes have been generated through covalent binding of lipid molecules either with native or recombinant S-layer proteins at nano-scale dimensions serving as “proof of concept” for the development of bio-sensing platform. Thus, in the light of benefits conferred by surface layer proteins for the development of highly efficient biosensors, an exciting path has been opened for broadening their translational applications in drug delivery, disease diagnosis, vaccines development, lab-on-chip devices etc. Therefore, this review intends to describe about the importance of surface layer proteins in the development of biosensors.

Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole(Cynoglossus semilaevis)

BMP2 plays crucial roles in vertebrate developmental process and acts as a bone inducer during osteogenesis. We present here the molecular cloning of bmp2 cDNA from the marine flatfish Cynoglossus semilaevis, and the analysis of bmp2 expression profiling and promoter function. The full length of bmp2 cDNA sequence is 2 048 bp,which encodes a protein of 422 amino acids. Tissue expression distribution of bmp2 was examined in 14 tissues of mature individuals by quantitative real time PCR（qRT-PCR）. The results revealed that bmp2 was expressed ubiquitously, and the highest expression level was detected in the spinal cord. Moreover, bmp2 expression levels were detected at 15 sampling time points of early developmental stages（egg, larva, juvenile and fingerling stages）.The highest expression level of bmp2 was observed at the gastrula stage, which was about ten times higher than those at the other three embryo stages. Whole-mount in situ hybridization showed that the bmp2 signal was strongly detected at the location of the crown-like larval fin, heart and liver, and slightly expressed in the notochord at one day post hatch（dph）; then the expression of bmp2 started to be concentrated in notochord at three dph. Subsequently, we characterized the 5′-flanking region of bmp2 by testing the promoter activity by Luciferase reporter assays. Positive regulatory region was detected at the location of –179 to ＋109. The predicted transcription factor binding sites（E-box binding factors, zinc finger transcription factor, etc.） in this region might participate in the transcriptional regulation of the bmp2 gene.

Gene Cloning and Expression Analysis of G Protein αq Subunit from Helicoverpa assulta (Guenée)

The cDNA encoding the G protein αq subunit was isolated from the antennae of Helicoverpa assulta （Guen6e） by reverse transcription polymerase chain reaction （RT-PCR） and named as HassGαq. Sequencing analysis showed that the fulllength of HassGαq open reading frame （ORF） is 1 062 bp, 353 amino acid residues are encoded. The predicted molecular weights （MW） and isoelectric point （PI） are 41.5 kD and 5.15, respectively. HassGαq gene was then constructed into expression vector pGEX-4T-2 for over expression in prokaryotic cells. The SDS-PAGE and Western blot analysis showed that induced by Isopropyl-β-D-Thiogalactoside （IPTG）, the GST-HassGαq fusion protein is expressed in Escherichia coil BL21, and its MW was found to be about 66 kD nearly equal to the predicted. In addition, RT-PCR analysis showed that the expressions of HassGαq are not tissue specific.

Guided Folding of Life’s Proteins in Integrate Cells with Holographic Memory and GM-Biophysical Steering

The current geometric and thermodynamic approaches in protein folding studies do not provide a definite solution to understanding mechanisms of folding of biological proteins. A major problem is that the protein is first synthesized as a linear molecule that subsequently must reach its native configuration in an extremely short time. Hydrophobicity-hydrophilicity models and random search mechanism cannot explain folding to the 3-D functional form in less than 1 second, as it occurs in the intact cell. We propose an integral approach, based on the embedding of proteins in the whole cellular context under the postulate: a life protein is never alone. In this concept the protein molecule is influenced by various long and short distance force fields of nature such as coherent electromagnetic waves and zero-point energy. In particular, the role of solitons is reviewed in relation to a novel GM-scale biophysical principle, revealed by us. This recent finding of a set of discrete EM frequency bands, that either promote or endanger life conditions, could be a key in further studies directed at the morphogenetic aspects of protein folding in a biological evolutionary context. In addition, an alternative hypothesis is presented in which each individual cell may store integral 3-D information holographically at the virtual border of a 4-D hypersphere that surrounds each living cell, providing a field receptive memory structure that is instrumental in guiding the folding process towards coherently oscillating protein networks that are crucial for cell survival.

玉米逆境胁迫响应基因ZmTPR1的表达和功能分析

在前期干旱-复水处理玉米转录组测序基础上,发现了一个响应干旱胁迫的基因ZmTPR1(Tetratricopeptide repeat 1),对该基因进行生物信息学分析,并探究其在不同组织及逆境胁迫下的表达模式,利用CRISPR-Cas9技术敲除拟南芥中的同源基因AtTPR1,分析干旱胁迫条件下纯合突变体植株的表型和生理生化指标,初步探究该基因的功能,为培育抗旱玉米品种提供基因资源。结果表明,ZmTPR1基因位于玉米的第3号染色体,编码421个氨基酸,具有保守的卷曲螺旋结构域,可能参与玉米对植物激素、干旱等胁迫的响应。ZmTPR1基因在玉米各组织中均表达,在幼茎中的表达量最高;干旱、高温、盐和缺氮胁迫均可诱导ZmTPR1基因的表达,干旱胁迫后表达量上调最明显;干旱胁迫后ZmTPR1基因在抗旱玉米自交系郑36中的表达量均极显著高于敏旱玉米自交系B73。敲除AtTPR1基因后拟南芥抗旱能力下降,Attpr1突变体在干旱胁迫下生长受到严重抑制,叶片萎蔫甚至干死,同时相对含水量、叶绿素含量、净光合速率及过氧化物酶和超氧化物歧化酶活性极显著降低,丙二醛含量极显著升高。综合来看,ZmTPR1基因参与玉米对多种非生物胁迫的响应过程,并在响应干旱胁迫中发挥着重要的作用。

新型细胞因子Metrnl与系统性红斑狼疮中巨噬细胞极化及其疾病活动度的关联研究

目的探讨镍纹样蛋白(Metrnl)在系统性红斑狼疮(SLE)及其严重并发症狼疮性肾炎(LN)患者中的水平,并探讨其与巨噬细胞极化状态及疾病活动度的关联。方法选取2023年6月至2024年5月该院收治的80例SLE患者作为研究对象,分为LN组(40例)和无LN组(40例),另选取同期该院30例年龄、性别匹配的健康体检者作为对照组。采用酶联免疫吸附试验检测Metrnl、M1型标志蛋白(iNOS)、M2型标志蛋白(Arg-1)、肿瘤坏死因子(TNF)-α和白细胞介素(IL)-10水平。采用Pearson相关分析SLE患者Metrnl水平与各项常规实验室指标水平,以及iNOS、Arg-1、TNF-α、IL-10的相关性。采用受试者工作特征(ROC)曲线分析Metrnl在SLE和LN中的诊断价值。结果LN组平均Metrnl水平为(98.35±37.12)pg/mL,明显低于无LN组的(167.89±25.74)pg/mL和对照组的(257.89±22.45)pg/mL,差异均有统计学意义(P<0.05)。Pearson相关分析结果显示,SLE患者Metrnl水平与TNF-α水平均呈负相关(r=-0.328,P=0.015),与Arg-1、IL-10水平均呈正相关(r=0.538、0.489,P<0.05)。ROC曲线分析结果显示,Metrnl诊断SLE患者和健康体检者的曲线下面积(AUC)为0.88,灵敏度为90.32%,特异度为76.00%;诊断无LN患者和LN患者的AUC为0.83,灵敏度为87.10%,特异度为73.53%。结论Metrnl在SLE及LN的免疫调控中发挥重要作用,可能通过影响巨噬细胞极化状态调节疾病活动度。SLE患者血清Metrnl水平与炎症因子等临床指标水平均有明显相关性,为其作为评估疾病活动度和治疗反应的潜在生物标志物提供了理论依据。

Gene Cloning and Tissue-Specific Expression of G Protein β Subunit in Microplitis mediator (Hymenoptera: Braconidae)

A gene encoding a novel G protein β subunit of β1 subclass, GβMmed was isolated from Microplitis mediator （Hymenoptera： Braconidae）. The full-length sequence of GβMmed is 1 119 bp, the cDNA contains a 1 023 bp open reading frame that encodes a protein with 340 amino acids, and the predicted molecular weight of GβMmed is 37.23 kDa and isoelectric point is 5.86. By the quantitative real-time RT-PCR method, the tissue-specific expression and quantitative changes in the developmental expression profile of GβMmed were detected. It was found that GβMmed was abundantly expressed in M. mediator antennae, head （without antennae）, thorax, abdomen, legs and the wings, and especially at high levels in abdomen. In antennae, expression varied through 1st day before emergence to 5-d-old adults, and had equal expression levels detected in females and males in total. In head, GβMmed expresses while initially high in females, and have another peaked in stage 4 and 1st day, in males showed a peak of GβMmed expression prior to emergence and relatively low levels after emergence. In female abdomen GβMmed expression levels have two peaks in stage 1 and the 5th d, but just have one peak in male abdomen in stage 1. In all other tissues expression was low and stable.

超声声脉冲辐射力成像-声触诊组织定量技术、丝氨酸/苏氨酸蛋白激酶基因检测与甲状腺弥漫性病变合并甲状腺癌中医证型的关系及诊断效能

目的分析超声声脉冲辐射力成像-声触诊组织定量技术(ARFI-VTQ)、B-Raf原癌基因丝氨酸/苏氨酸蛋白激酶(BRAF)基因检测对甲状腺弥漫性病变合并甲状腺癌的诊断效能,以及与甲状腺癌中医证型的关系。方法选择2023年1月至2023年12月于河北省沧州中西医结合医院超声医学一科进行甲状腺弥漫性病变合并甲状腺结节检查的患者130例作为研究对象,根据病理检查结果分为良性组(n=88)和恶性组(n=42),所有患者均行超声ARFI-VTQ检查和BRAF基因检测,并进行中医辨证分型。比较良性组与恶性组、恶性组不同中医证型患者常规超声特征、SWV值以及BRAF基因检测阳性率;行受试者工作特征(ROC)曲线分析,分析超声ARFI-VTQ和BRAF基因检测对甲状腺弥漫性病变合并甲状腺癌的诊断效能。结果良性组与恶性组患者,以及恶性组气滞血瘀证、肝郁痰凝证、气阴两虚证患者常规超声特征的边界、形状、回声和内部血流比较差异均无统计学意义(P>0.05)。恶性组SWV值高于良性组(P<0.05);恶性组不同中医证型患者SWV值也存在明显差异,其中气滞血瘀证最高,气阴两虚证最低,比较差异有统计学意义(P<0.05)。良性组BRAF基因突变阳性率低于恶性组(P<0.05);恶性组不同中医证型患者BRAF基因突变阳性率比较差异无统计学意义(P>0.05)。ROC曲线分析结果显示,SWV值和BRAF基因检测结果对甲状腺弥漫性病变合并甲状腺癌的曲线下面积(AUC)分别为0.759和0.674,敏感度分别为88.10%和76.19%,特异性分别为90.17%和84.97%,约登指数分别为0.783和0.612。但是联合检测降低了检测的特异性,导致约登指数降低为0.369。结论超声ARFI-VTQ检查、BRAF基因检测对甲状腺弥漫性病变合并甲状腺癌的诊断具有重要的价值,其中ARFI-VTQ检查对甲状腺弥漫性病变合并甲状腺癌患者的辨证分型有重要的参考价值。

黄曲条跳甲短链气味结合蛋白基因PstrOBP-sc分子和功能特点分析

【目的】分析黄曲条跳甲(Phyllotreta striolata)短链气味结合蛋白(Odorant binding protein,OBP)基因PstrOBP-sc分子和功能特点,为揭示昆虫短链OBP基因的功能提供参考。【方法】利用逆转录聚合酶链反应(RT-PCR)克隆黄曲条跳甲短链Classic OBP基因,并对其进行生物信息学分析;通过逆转录聚合酶链反应技术分析短链OBP基因在黄曲条跳甲雌、雄成虫不同组织中的表达情况;构建重组表达载体异源表达短链OBP基因,以聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blotting)验证目的蛋白;运用荧光结合试验检测短链OBP的配体结合能力。【结果】通过逆转录聚合酶链反应克隆验证,成功获得1条具有完整开放阅读框的黄曲条跳甲短链OBP基因PstrOBP-sc。PstrOBP-sc基因含有1个357 bp的开放阅读框,编码118个氨基酸残基,N末端具有1个由20个氨基酸残基组成的信号肽,成熟蛋白PSTROBP-SC仅由98个氨基酸残基组成,序列中含有6个保守的半胱氨酸,属于典型短链OBP基因。蛋白二级结构预测结果显示,PSTROBP-SC蛋白仅存在5个α-螺旋,且在第5个α-螺旋的下游仅有2个氨基酸残基。PstrOBP-sc基因组织表达模式及异源表达的PSTROBP-SC蛋白配体结合能力研究结果表明,PstrOBP-sc基因在黄曲条跳甲雌、雄成虫的所有供试组织中均有表达,而非特异性表达于嗅觉器官。同时,异源表达PSTROBP-SC不能结合荧光探针1-NPN。【结论】PstrOBP-sc基因的分子和功能特点不同于当前广泛研究的中长链OBP基因,其在生物体内很可能行使嗅觉以外的其他生理功能。

Organizing and Disorganizing Resonances of Microtubules, Stem Cells, and Proteins Calculated by a Quantum Equation of Coherence

Conformational states of microtubules and proteins have typical spatial-spectral arrangements of atoms, called spatial coherence, that are characteristic for building, homeostasis, decay, and apoptosis. Microtubules show a principle of a self-organizing-synergetic structure called a Fröhlich-Bose-Einstein state. The spatial coherence of this state can be described by a toroidal quantum equation of coherence. In this space, microtubules and proteins have typical discrete frequency patterns. These frequencies comply with two proposed quantum wave equations of respective coherence (regulation) and decoherence (deregulation), that describe quantum entangled and disentangled states. The proposed equation of coherence shows the following typical scale invariant distribution of energy: En = ħωref 2q3m. The proposed model supports quantum entanglement and is in line with the earlier published models of Fröhlich, Davydov, and Chern. A meta-analysis shows a semi-harmonic scale-invariant pattern for microtubules, stem cells, proteins, and EEG- and MEG-patterns. A fit has been found for about 50 different organizing frequencies and 5 disorganizing frequencies of measured microtubule frequencies that fit with the calculated values of the proposed quantum equations, which are positioned in a nested toroidal geometry. All measured and analysed frequencies of microtubules comply with the same energy distribution found for Bose-Einstein condensates. The overall results show a presence of an informational quantum code, a direct relation with the eigenfrequencies of microtubules, stem cells, DNA, and proteins, that supplies information to realize biological order in life cells and substantiates a collective Fröhlich-Bose-Einstein type of behaviour and further support the models of Tuszynski, Hameroff, Bandyopadhyay, Del Giudice and Vitiello, Katona, Pettini, and Pokorny.