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Non-Hodgkin's Lymphoma (NHL) remains a significant challenge in hematology, with chemotherapy and radiation therapy as conventional treatment options, albeit with limitations such as adverse effects. Immunotherapy, particularly bispecific antibodies (BsAbs) T cell engagers (TCEs), has emerged as a promising approach. Despite their potential, TCEs pose challenges, including adverse events like cytokine release syndrome. Understanding the structural details of TCEs and their interactions with target proteins is crucial for optimizing their therapeutic efficacy and toxicity. In this study, we further developed our protocol MCCS-Docker for protein-protein interactions and applied it to investigate the structural intricacies of CD3 interactions with therapeutic antibodies such as OKT3, UCHT1, Mosunetuzumab, Odronextumab, Glofitamab, and Epcoritamab using computational modeling techniques. Our analysis not only approved the effectiveness of our updated MCCS-Docker protocol but also revealed detailed binding interactions between the BsAbs and CD3, elucidating key residues of Tyrosine and Asparagine in the antibodies involved in the binding interface. Molecular dynamics simulations validated the stability of these interactions over time, confirming the reliability of the binding poses generated from docking studies. Overall, our study offered a novel method to predict critical residues in protein-protein interactions and enhanced the understanding of the structural determinants governing BsAb interactions with target proteins, offering valuable insights for designing and optimizing immunotherapeutic agents for NHL and related hematologic malignancies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

MiRNA-21 is recognized as an important biological marker for the diagnosis, treatment, and prognosis of breast cancer. Here, we have created a nanochannel biosensor utilizing the duplex-specific nuclease (DSN) signal amplification strategy to achieve the detection of miRNAs. In this system, DNA as the capture probe was covalently immobilized on the surface of nanochannels, which hybridized with the target miRNA and forms RNA/DNA duplexes. DSN could cleave the probe DNA in RNA/DNA duplexes, recycling target miRNA, which may again hybridized with other DNA probes. After N cycles, most of the DNA probes had been cleaved, and the content of miRNA could be quantified by detecting changes in surface charge density. This biosensor can distinguish miR-21 from non-complementary miRNAs and one-base mismatched miRNAs, with reliable detection limits as low as 1 fM in PBS. In addition, we had successfully applied this method to analysis of total RNA samples in MCF-7 cells and HeLa cells, and the nanochannels had also shown excellent responsiveness and strong anti-interference ability. This new method is expected to contribute to miRNA detection in clinical diagnostics, providing a unique approach to detecting and distinguishing disease-associated molecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Tumor-derived exosome (TD-Ex) serves as a crucial early diagnostic biomarker of pancreatic cancer (PC). However, accurate identification of TD-Ex from PC is still a challenging work. In this paper, a detection microsystem that integrates magnetic separation and FET biosensor is developed, which is capable of selectively separating TD-Ex of PC from the plasma and detecting exosomal miRNA10b in a sensitive and specific manner. The magnetic beads were functionalized with dual antibody (GPC-1 antibody and EpCAM antibody), enabling selective recognition and capture of PC-derived exosomes. On the other hand, a peptide nucleic acid (PNA)- functionalized reduced graphene oxide field-effect transistor (RGO FET) biosensor was subsequently utilized to detect the exosomal miRNA10b, which is highly expressed in PC- derived exosomes. This system could achieve a low detection limit down to 78 fM, and selectively identify miRNA10b from single-base mismatched miRNA. In addition, 40 clinical plasma samples were tested with this microsystem, and the results indicate that it could effectively distinguish PC patients from healthy individuals. The assay combines specific capture and enrichment of PC-derived exosomes with sensitive and selective detection of exosomal miRNA, showing its potential to be used as an effective scheme for PC early diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Right atrial (RA) structural and functional evaluations have recently emerged as powerful biomarkers for adverse events in various cardiovascular conditions. Quantitative analysis of the right atrium, usually performed with volume changes or speckle-tracking echocardiography (STE), has markedly changed our understanding of RA function and remodeling. Knowledge of reference echocardiographic values and measurement methods of RA volumes and myocardial function is a prerequisite to introduce RA quantitation in the clinical routine. This review describes the methodology, benefits and pitfalls of measuring RA size and function by echocardiography based on the current understanding of right atrial anatomy and physiological function and provides the current knowledge of right atrial function in related cardiac diseases., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Fluctuation disturbance of organic loading rate (OLR) is common in actual anaerobic digestion (AD), but its effects on AD of municipal sludge gets little attention. This study investigated the responses of reactor performance and active microbial community in mesophilic and thermophilic AD of municipal sludge before, during and after OLR periodic fluctuation disturbance. The performance of both reactors were similar before and after disturbance although some parameter values changed during the disturbance, which indicated their enough buffer capacity to OLR periodic fluctuation. Different microbial community at RNA level was observed in the two reactors. When the OLR disturbance commenced, the microbial community changed greatly in thermophilic AD. Error and attack tolerance of the microbial network was analyzed in order to learn the response mechanisms to OLR disturbance. The results assisted that the thermophilic microbial community was more vulnerable, but the reactor performance of which could be maintained using the functional redundancy strategy under OLR fluctuation disturbance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This paper proposes a new control strategy to deal with three different types of uncertainties for a class of time-varying delay nonlinear systems. Quite different from related celebrated works, the considered systems allow the existence of parameter uncertainties in the output function and nonlinearities, dynamic uncertainties of the unmeasurable state, and continuous external disturbances, which invokes the motivation of the paper. The objective is to construct a continuous output feedback controller by utilizing a new restructured integral function and the double-domination method. The novelty control design is the capability of tackling zero-dynamic and unknown output function simultaneously, thereby guaranteeing the state convergence of the closed-loop system. Finally, the proposed scheme is applied to a practical example and a numerical one simultaneously to demonstrate the effectiveness and the superiority., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 ISA. Published by Elsevier Ltd. All rights reserved.)

Titanium (Ti) and its alloys are believed to be promising scaffold materials for dental and orthopedic implantation due to their ideal mechanical properties and biocompatibility. However, the host immune response always causes implant failures in the clinic. Surface modification of the Ti scaffold is an important factor in this process and has been widely studied to regulate the host immune response and to further promote bone regeneration. In this study, a calcium-strontium-zinc-phosphate (CSZP) coating was fabricated on a Ti implant surface by phosphate chemical conversion (PCC) technique, which modified the surface topography and element constituents. Here, we envisioned an accurate immunomodulation strategy via delivery of interleukin (IL)-4 to promote CSZP-mediated bone regeneration. IL-4 (0 and 40 ng/mL) was used to regulate immune response of macrophages. The mechanical properties, biocompatibility, osteogenesis, and anti-inflammatory properties were evaluated. The results showed that the CSZP coating exhibited a significant enhancement in surface roughness and hydrophilicity, but no obvious changes in proliferation or apoptosis of bone marrow mesenchymal stem cells (BMMSCs) and macrophages. In vitro, the mRNA and protein expression of osteogenic related factors in BMMSCs cultured on a CSZP coating, such as ALP and OCN, were significantly higher than those on bare Ti. In vivo, there was no enhanced bone formation but increased macrophage type 1 (M1) polarization on the CSZP coating. IL-4 could induce M2 polarization and promote osteogenesis of BMMSCs on CSZP in vivo and in vitro. In conclusion, the CSZP coating is an effective scaffold for BMMSCs osteogenesis, and IL-4 presents the additional advantage of modulating the immune response for bone regeneration on the CSZP coating in vivo., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bioglass scaffolds have great application potentials in orthopedics, and Ursolic acid (UA) can effectively promote in vivo new bone formation. Herein, we for the first time developed the mesoporous bioglass/chitosan porous scaffolds loaded with UA (MBG/CS/UA) for enhanced bone regeneration. The MBG microspheres with particle sizes of ~300 nm and pore sizes of ~3.9 nm were uniformly dispersed on the CS films. The mesoporous structure within the MBG microspheres and the hydrogen bonding between the scaffolds and UA drugs made the MBG/CS/UA scaffolds have controlled drug release performances. The as-released UA drugs from the scaffolds increased remarkably the alkaline phosphatase activity, osteogenic differentiation related gene type I collagen, runt-related transcription factor 2 expression, and osteoblast-associated protein expression. Moreover, the results of micro-CT images, histomorphological observations demonstrated that the MBG/CS/UA scaffolds improved new bone formation ability. Therefore, the MBG/CS/UA porous scaffolds can be used as novel bone tissue engineering materials., (Copyright © 2018. Published by Elsevier Inc.)

Background: Antiplatelet therapies for secondary prevention of ischemic stroke or transient ischemic attack (TIA) is a highly active research topic with five critical drugs obtained by visual analysis. We aimed to compare and rank multiple antiplatelet therapies using a network meta-analysis., Methods: Relevant medical databases were searched. Eligible randomized controlled trials (RCTs) which examined any comparisons involving mono- or dual antiplatelet therapies, based on aspirin, clopidogrel, dipyridamole, ticlopidine, cilostazol and placebo for patients with noncardioembolic ischemic stroke or TIA, were included. 14 outcomes were assessed. Primary outcomes were stroke recurrence, composite events (stroke recurrence, myocardial infarction and vascular death), and intracranial hemorrhage. PROSPERO registered number CRD42017069728., Results: 45 RCTs with 173,131 patients were included in network meta-analysis, involving eight antiplatelet therapies. Cilostazol and clopidogrel were statistically more efficacious than aspirin (odds ratio (OR) = 0.64, 95% confidence interval (CI) = 0.47-0.88; OR = 0.77, 95%CI = 0.62-0.95) and dipyridamole (OR = 0.64, 95%CI = 0.44-0.93; OR = 0.76, 95%CI = 0.58-0.99) in reducing stroke recurrence, and showed significant benefits in reducing composite events compared with aspirin (OR = 0.63, 95%CI = 0.45-0.89; OR = 0.90, 95%CI = 0.83-0.97). No significant difference was found between cilostazol and clopidogrel in intracranial hemorrhage. Weighted regression suggested cilostazol was hierarchically the optimum treatment in consideration of both efficacy and safety, followed by clopidogrel., Conclusion: Cilostazol and clopidogrel are probably promising options for secondary prevention of ischemic stroke or TIA. Both of them reduce stroke recurrence similarly compared with aspirin or dipyridamole, and reduce composite events compared with aspirin. Further studies are needed to confirm this finding., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: To compare the image quality of navigator-triggered (NT) 3D MR cholangiopancreatography (MRCP) with and without a patient-adapted respiratory training, in clinical patients with painful pancreatobiliary disorders., Materials and Methods: With institutional review board approval, hospitalized patients with painful pancreatobiliary disorders who were scheduled for MRCP study were prospectively enrolled. The numerical rating scale (NRS) of abdominal pain during the examination was recorded. Special patient-adapted respiratory training was conducted before the examination. A control group of patients was enrolled with the same criteria, who received ordinary instructions only (n=60 for each group). A subgroup of patients (n=10) underwent MRCP studies with ordinary instructions first and with patient-adapted training later. Acquisition time was recorded. General image quality, degree of artifacts and visualization of 12 segments of the pancreatobiliary tree were rated on a five-point scale and compared between the groups., Result: Both groups had similar NRS of pain. There was a significant improvement in image quality (p<0.01) as well as visualization of right posterior hepatic duct (p=0.045), left lateral hepatic duct (p=0.037), and pancreatic duct (p<0.01 for head, body and tail segments) in patients receiving respiratory training. The other segments showed no significant differences. The percentage of patients with severe and extensive imaging artifacts decreased from 18.3%(11/60) to 8.3%(5/60). The acquisition time was shorter (175±54s vs 249±67s, p<0.01) in patients with respiratory training., Conclusion: Patient-adapted respiratory training improves the image quality of NT-MRCP in patients with painful pancreatobiliary disorders., (Copyright © 2017. Published by Elsevier Inc.)

There are few data on the molecular epidemiology of cryptococcosis in China. Here we investigated the species distribution, molecular types and antifungal susceptibilities of 312 Cryptococcus neoformans species complex isolates from ten hospitals over 5 years. Isolates were identified by internal transcribed spacer (ITS) sequencing and by two matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems. Multilocus sequence typing (MLST) was used to verify species/variety and to designate molecular types. Susceptibility to six antifungal drugs was determined by the Sensititre YeastOne™ method. Cryptococcus neoformans was the predominant species (305/312 isolates (97.8%), all were ITS type 1, serotype A), of which 89.2% (272/305) were C. neoformans var. grubii MLST sequence type (ST) 5 and 6.2% (19/305) were ST31. Other C. neoformans var. grubii STs were rare but included six novel STs. Only two strains were C. neoformans var. neoformans (both serotype AD). Cryptococcus gattii was uncommon (n = 7, four ITS types) and comprised five MLST STs including one novel ST. For C. neoformans var. grubii, the proportion of isolates with non-wild-type MICs to fluconazole significantly rose in the fourth study year (from 0% (0/56 isolates) in the first year to 23.9% (17/71) in the fourth year), including five isolates with fluconazole MICs of ≥32 mg/L. The study has provided useful data on the species epidemiology and their genetic diversity and antifungal susceptibility. The proportional increase in isolates with non-wild-type MICs to fluconazole is noted., (Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

With the aim of gathering temporal trends on bacterial epidemiology and resistance from multiple laboratories in China, the CHINET surveillance system was organized in 2005. Antimicrobial susceptibility testing was carried out according to a unified protocol using the Kirby-Bauer method or automated systems. Results were analyzed according to Clinical and Laboratory Standards Institute (CLSI) 2014 definitions. Between 2005 and 2014, the number of bacterial isolates ranged between 22,774 and 84,572 annually. Rates of extended-spectrum β-lactamase production among Escherichia coli isolates were stable, between 51.7 and 55.8%. Resistance of E. coli and Klebsiella pneumoniae to amikacin, ciprofloxacin, piperacillin/tazobactam and cefoperazone/sulbactam decreased with time. Carbapenem resistance among K. pneumoniae isolates increased from 2.4 to 13.4%. Resistance of Pseudomonas aeruginosa strains against all of antimicrobial agents tested including imipenem and meropenem decreased with time. On the contrary, resistance of Acinetobacter baumannii strains to carbapenems increased from 31 to 66.7%. A marked decrease of methicillin resistance from 69% in 2005 to 44.6% in 2014 was observed for Staphylococcus aureus. Carbapenem resistance rates in K. pneumoniae and A. baumannii in China are high. Our results indicate the importance of bacterial surveillance studies., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

HIV-1 (human immunodeficiency virus type 1) uses its trimeric gp160 envelope (Env) protein consisting of non-covalently associated gp120 and gp41 subunits to mediate entry into human T lymphocytes. A facile virus fusion mechanism compensates for the sparse Env copy number observed on viral particles and includes a 22-amino-acid, lentivirus-specific adaptation at the gp41 base (amino acid residues 662-683), termed the membrane proximal external region (MPER). We show by NMR and EPR that the MPER consists of a structurally conserved pair of viral lipid-immersed helices separated by a hinge with tandem joints that can be locked by capping residues between helices. This design fosters efficient HIV-1 fusion via interconverting structures while, at the same time, affording immune escape. Disruption of both joints by double alanine mutations at Env positions 671 and 674 (AA) results in attenuation of Env-mediated cell-cell fusion and hemifusion, as well as viral infectivity mediated by both CD4-dependent and CD4-independent viruses. The potential mechanism of disruption was revealed by structural analysis of MPER conformational changes induced by AA mutation. A deeper acyl chain-buried MPER middle section and the elimination of cross-hinge rigid-body motion almost certainly impede requisite structural rearrangements during the fusion process, explaining the absence of MPER AA variants among all known naturally occurring HIV-1 viral sequences. Furthermore, those broadly neutralization antibodies directed against the HIV-1 MPER exploit the tandem joint architecture involving helix capping, thereby disrupting hinge function., (© 2013.)

Unlabelled: Septicemia is a common cause of morbidity and mortality among newborns in the developing world. However, accurate clinical diagnosis of neonatal sepsis is often difficult because symptoms and signs are often nonspecific. Blood culture has been the gold standard for confirmation of the diagnosis. However, the sensitivity is low and results are usually not promptly obtained. Therefore, the diagnosis of sepsis is often based on clinical signs in association with laboratory tests such as platelets count, immature/total neutrophils ratio (I/T), and a rise in C-reactive protein (CRP). Polymerase chain reaction (PCR) methods for the detection of neonatal sepsis represent new diagnostic tools for the early identification of pathogens., Methods: During a 4-month prospective study, 16S rRNA PCR was compared with conventional blood culture for the diagnosis of neonatal bacterial sepsis. In addition, the relationship between known risk factors, clinical signs, laboratory parameters, and the diagnosis of sepsis was considered., Results: Sepsis was suspected in 706 infants from the intensive neonatal care unit. They all were included in the study. The number of positive cultures and positive PCR results were 95 (13.5%) and 123 (17.4%), respectively. Compared with blood culture, the diagnosis of bacterial sepsis by PCR revealed a 100.0% sensitivity, 95.4% specificity, 77.2% positive predictive value, and 100.0% negative predictive value. In this study, Apgar scores at 5 min, weight, icterus, irritability, feeding difficulties, gestational age (GA), premature rupture of membrane (PRM), platelets count, I/T, and a marked rise in CRP were important in establishing the diagnosis of sepsis in the newborn. In addition, weight, GA, PRM, irritability, duration of antibiotic usage, mortality rate, and number of purulent meningitis cases were significantly different between early-onset sepsis and late-onset sepsis., Conclusion: 16S rRNA PCR increased the sensitivity in detecting bacterial DNA in newborns with signs of sepsis, allowed a rapid detection of the pathogens, and led to shorter antibiotic courses. However, uncertainty about the bacterial cause of sepsis was not reduced by this method. 16S rRNA PCR needs to be further developed and improved. Blood culture is currently irreplaceable, since pure isolates are essential for antimicrobial drug susceptibility testing., (Copyright © 2013. Published by Elsevier SAS.)

The transforming growth factor-β (TGF-β) signaling pathway promotes tissue fibrosis and scarring through SMAD (small mothers against decapentaplegic)-dependent and SMAD-independent mechanisms. However, inhibition of SMAD-mediated signal transduction alone induces an excessive inflammatory response that impairs the antifibrotic effects of TGF-β inhibitors. In this study, we designed and characterized a dual-functional transcription activator protein 1 (AP-1) and SMAD decoy oligodeoxynucleotide, antifibrosis oligodeoxynucleotide 4 (AFODN4) in vitro and in vivo. AFODN4 binds directly to recombinant AP-1 and SMAD with high affinity. AFODN4 significantly inhibited the DNA-binding and transcriptional activities of both AP-1 and SMAD, as well as the production of fibrotic mediators stimulated by TGF-β1 or TGF-β2 in L929 murine fibroblasts. Local administration of AFODN4 significantly inhibited fibrosis associated with acute dermal wounds in mice. Intriguingly, AFODN4 inhibited AP-1-mediated production of proinflammatory mediators, which can be caused by blockage of SMAD alone in vitro and in vivo. Collectively, these findings suggest that dual inhibition of SMAD and AP-1 signaling by AFODN4 is a useful strategy for the development of new antifibrotic agents.

Continuous exposure of oocytes to elevated concentrations of insulin compromises embryonic developmental competence. However, the effects of insulin on oogenesis from fetal germ cells are unknown. The objective of this study was to assess the effect of continuous insulin exposure, with or without FSH, on oogenesis and follicular development. A simple and efficient method was established that could be used to obtain oocytes from pre-meiotic germ cells in 12.5days post-coitum (dpc) fetal mouse ovaries using a three-dimensional culture system with serum-free medium. Mouse 12.5dpc fetal ovaries were cultured for 14days with or without insulin/FSH. Low (0.2-1microg/ml) or high (5-20microg/ml) doses of insulin retarded oocyte growth in vitro. Insulin at 5microg/ml led to significant oocyte growth retardation (P<0.05), while FSH alleviated the deleterious effect of insulin. Most importantly, the proportion of secondary follicles at 12days post-culture in the presence of insulin was reduced significantly compared with controls (P<0.05). Expression levels of genes specific for ovarian cells, e.g. Cx37, Cx43, Scp3, Bax and FSHR, were significantly reduced when exposed to insulin during oogenesis (P<0.05). The data suggest that insulin has a profound detrimental effect on oogenesis and folliculogenesis in vitro., (Copyright (c) 2009 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

It has been reported that penile PDE5 expression was under androgen regulation. However it remained unknown whether the observed change in PDE5 expression in castrated animals was under direct androgen regulation or due to changes in smooth muscle content. In the present study we showed that castration of rats caused a reduction of penile size and cavernous smooth muscle content. Immunostaining detected concomitant reduction of PDE5 and alpha smooth muscle actin (alpha-SMA) expression in the corpus cavernosum of castrated rats. Real-time PCR and Western blotting detected no change of PDE5 expression when normalized with alpha-SMA expression in castrated rats. Androgen receptor (AR) expression was increased while PDE5 expression remained unchanged in DHT-treated rat cavernous smooth muscle cells (CSMC). Prostate specific antigen (PSA) promoter activity was upregulated while PDE5A promoter activity remained unchanged in DHT-treated CSMC. Thus, PDE5 expression was not under direct androgen regulation.

Recruitment of signaling molecules to the cytoplasmic domains of the CD3 subunits of the T-cell receptor (TCR) is crucial for early T-cell activation. These transient associations either do or do not require tyrosine phosphorylation of CD3 immune tyrosine activation motifs (ITAMs). Here we show that the non-ITAM-requiring adaptor protein Nck forms a complex with an atypical PxxDY motif of the CD3epsilon tail, which encompasses Tyr166 within the ITAM and a TCR endocytosis signal. As suggested by the structure of the complex, we find that Nck binding inhibits phosphorylation of the CD3epsilon ITAM by Fyn and Lck kinases in vitro. Moreover, the CD3epsilon-Nck interaction downregulates TCR surface expression upon physiological stimulation in mouse primary lymph node cells. This indicates that Nck performs an important regulatory function in T lymphocytes by inhibiting ITAM phosphorylation and/or removing cell surface TCR via CD3epsilon interaction.

The aim of this study was to investigate the relationship between 16-slice spiral CT perfusion imaging and tumor angiogenesis and cyclin D1 expression in patients with peripheral lung cancer. Fifty-eight patients with peripheral lung cancer underwent 16-slice spiral CT perfusion imaging. The CT perfusion imaging was analyzed for time density curve (TDC), perfusion parametric maps, and the respective perfusion parameters. Correlation between the respective perfusion parameters and immunohistochemical findings of microvessel density measurement and cyclin D1 expression was evaluated.

Non-uniform sampling is shown to provide significant time savings in the acquisition of a suite of three-dimensional NMR experiments utilized for obtaining backbone assignments of H, N, C', CA, and CB nuclei in proteins : HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, and HN(CO)CACB. Non-uniform sampling means that data were collected for only a subset of all incremented evolution periods, according to a user-specified sampling schedule. When the suite of six 3D experiments was acquired in a uniform fashion for an 11 kDa cytoplasmic domain of a membrane protein at 1.5 mM concentration, a total of 146 h was consumed. With non-uniform sampling, the same experiments were acquired in 32 h and, through subsequent maximum entropy reconstruction, yielded spectra of similar quality to those obtained by conventional Fourier transform of the uniformly acquired data. The experimental time saved with this methodology can significantly accelerate protein structure determination by NMR, particularly when combined with the use of automated assignment software, and enable the study of samples with poor stability at room temperature. Since it is also possible to use the time savings to acquire a greater numbers of scans to increase sensitivity while maintaining high resolution, this methodology will help extend the size limit of proteins accessible to NMR studies, and open the way to studies of samples that suffer from solubility problems.

The heterophilic CD2-CD58 adhesion interface contains interdigitating residues that impart high specificity and rapid binding kinetics. To define the hot spot of this counter-receptor interaction, we characterized CD2 adhesion domain variants harboring a single mutation of the central Tyr86 or of each amino acid residue forming a salt link/hydrogen bond. Alanine mutations at D31, D32 and K34 on the C strand and K43 and R48 on the C' strand reduce affinity for CD58 by 47-127-fold as measured by isothermal titration calorimetry. The Y86A mutant reduces affinity by approximately 1000-fold, whereas Y86F is virtually without effect, underscoring the importance of the phenyl ring rather than the hydroxyl moiety. The CD2-CD58 crystal structure offers a detailed view of this key functional epitope: CD2 D31 and D32 orient the side-chain of CD58 K34 such that CD2 Y86 makes hydrophobic contact with the extended aliphatic component of CD58 K34 between CD2 Y86 and CD58 F46. The elucidation of this hot spot provides a new target for rational design of immunosuppressive compounds and suggests a general approach for other receptors., (Copyright 2001 Academic Press.)

The CD3 polypeptides (epsilon, gamma, and delta) are non-covalently associated signaling subunits of the T cell receptor which form non-disulfide linked epsilongamma and epsilondelta heterodimers. With the goal of investigating their structure, Escherichia coli expression was utilized to produce CD3 ectodomain fragments including the murine CD3epsilon subunit N-terminal Ig-like extracellular domain alone or as a single chain construct with that of CD3gamma. The latter links the CD3gamma segment to the C terminus of the CD3epsilon segment via a 26 amino acid peptide (scCD3epsilongamma26). Although CD3epsilon could be produced at high yield when directed to inclusion bodies, the refolded monomeric CD3epsilon was not native as judged by monoclonal antibody binding using surface plasmon resonance and was largely unstructured by (15)N-(1)H two-dimensional NMR analysis. In contrast, scCD3epsilongamma26 could be refolded readily into a native state as shown by CD, NMR and mAb reactivity. The linker length between CD3epsilon and CD3gamma is critical since scCD3epsilongamma16 containing a 16 residue connector failed to generate a stable heterodimer. Collectively, the results demonstrate that: (i) soluble heterodimeric fragments of CD3 can be produced; (ii) cotranslation of CD3 chains insures proper folding even in the absence of the conserved ectodomain stalk region (CxxCxE); and (iii) CD3epsilon has a more stable tertiary protein fold than CD3gamma., (Copyright 2000 Academic Press.)