The Earliest Discovery of the Role of Magnesium Ions on Stabilizing the Tertiary Structure of the Transfer RNA and Its Biological Significance —A Short Memoir

In early of 1960s, I was a graduate student studying on tRNA biochemistry. In the course of the research, the magnesium ions stabilized the tertiary structure of tRNA, resulting in its resistance to enzymatic degradation was discovered independently. The experiment of deaminated (denatured) tRNA obtained from native tRNA was designed and conducted and further proved the validity of this finding. It was found that magnesium ions could stabilize the tertiary structure of the natrive tRNA but could not stabilize structure of the deaminated tRNA. In term of the methodology, this stabilization technique has been widely applied in sequencing analysis of RNA and has greatly promoted the progress in the study of primary structure of RNA. More importantly, the stabilization of the tertiary structure of RNA by magnesium ions plays a key role both in the processing of messenger RNAs and the ribozyme activity. After our first article in Chinese was published in 1963, a paper of Nishimura & Novelli came into our note. The received date of their paper was March 22 of 1963, only 4 days earlier than that of our first paper. Thus, we and Nishimura & Novelli made almost at the same time the earliest discovery of the role of magnesium ions on stabilizing the tertiary structure of the transfer RNA and thus resulted in resistance of tRNA degradation by enzymes. However, this discovery was not initially appreciated for a period of time but was finally “visualized” and proved by X-ray crystal structure of yeast phenylalanine tRNA, which has provided more accurate information on the geometry of the magnesium-binding sites in tRNA.

Tertiary structure-based protein classificationby virtual-bond-angles series

Structure-based protein classification can be based on the similarities in primary, second or tertiary structures of proteins. A method using virtual-bond-angles series that transformed the protein space configuration into a sequence was used for the classification of three-dimensional structures oi proteins. By transforming the main chains formed by C^a atoms of proteins into sequences, the series of virtual-bond-angles corresponding to the tertiary structure of the proteins were constructed. Then a distance-based hierarchical clustering method similar to Ward method was introduced to classify these virtual-bond-angles series of proteins. 200 files of protein structures were selected from Brookheaven protein data bank, and 11 clusters were classified.

Insights into tertiary lymphoid structures in the solid tumor microenvironment: anti-tumor mechanism, functional regulation, and immunotherapeutic strategies

Tertiary lymphoid structures(TLSs)are ectopic immune cell aggregations that develop in peripheral tissues in response to a wide range of chronic inflammatory conditions,including infection,autoimmune disease,and cancer.In the tumor microenvironment(TME),the structures of TLSs,including B-cell-and T-cell-enriched areas indicate that the TLSs might be the local site during the initiation and maintenance of humoral and cellular immune responses against cancers.Numerous studies have evaluated the expression of TLSs in different cancer patients and their association with prognoses of cancer patients.It was shown that welldeveloped TLSs characterized by mature B cells synthesized tumor specific antibodies,which were considered as specific markers for a good prognosis.However,there are still some immunosuppressive factors existing in the TLSs that may affect anti-tumor responses.These factors include dysfunctional B cells,regulatory T cells,and T follicular regulatory cells.The complexity and heterogeneity of the TLS composition may affect the function and activity of TLSs;it is therefore essential to fully understand the function and influencing factors in TLSs.It has been reported that checkpoint inhibitors and vaccines are currently being developed to reprogram the TME by establishing mature TLSs to improve cancer immunotherapies.In this review,we focused on recent advances in TLSs in human solid tumors,including structural characteristics and classes,antitumor mechanisms,immunosuppressive factors,and TLSbased therapeutic approaches.

Tertiary lymphoid structure patterns predicted anti-PD1 therapeutic responses in gastric cancer

Objective:Recent studies have highlighted the distinct value of tertiary lymphoid structure(TLS)for immunotherapeutic response prediction.However,it remains unclear whether TLS could play such roles in gastric cancer(GC).Methods:In this study,tumor tissue slices from 292 GC patients from Zhongshan Hospital were firstly reviewed to explore the correlation between TLS and clinical characteristics.Subsequently,we curated 38 reported genes that may function as triggers of TLS and performed consensus molecular subtyping in public RNA-seq datasets to determine TLS patterns in GC.Based on the differentially expressed genes acquired from two TLS patterns,we quantified TLS-related genes on the principal component analysis(PCA)algorithm to develop TLS score.A Zhongshan immunotherapy cohort including 13 patients who received programmed cell death 1(PD1)blockade therapy was established to conduct RNA sequencing analysis and multiplex immunohistochemistry(mIHC)tests using formalin-fixed and paraffin-embedded(FFPE)tissues.The corresponding TLS score and immune cell counts were further compared based on therapeutic response variations.Results:Mature TLS was revealed as an independent prognostic factor in 292 GC patients.Patients with higher TLS score was characterized by prolonged survival time and superior response to immunotherapy.TLS score was correlated with immunotherapy-related characters,such as microsatellite instability(MSI)and tumor mutation burden(TMB).In addition,RNA-seq data analysis in the Zhongshan immunotherapy cohort indicated that a higher TLS score was correlated with a superior response to PD1 blockade therapy.mIHC tests also revealed that PD1+CD8+T cell counts were significantly increased in the high-TLS score group.Conclusions:This study highlighted that TLS was significantly associated with immune landscape diversity and complexity.Quantitatively evaluating TLS patterns of individual tumor will strengthen our understanding of TME characteristics and promote more effective immunotherapy strategies.

Detection and quantitative analysis of tumor-associated tertiary lymphoid structures

Tumor-associated tertiary lymphoid structures(TLSs)are ectopic lymphoid formations within tumor tissue,with mainly B and T cell populations forming the organic aggregates.The presence of TLSs in tumors has been strongly associated with patient responsiveness to immunotherapy regimens and improving tumor prognosis.Researchers have been motivated to actively explore TLSs due to their bright clinical application prospects.Various studies have attempted to decipher TLSs regarding their formation mechanism,structural composition,induction generation,predictive markers,and clinical utilization.Meanwhile,the scientific approaches to qualitative and quantitative descriptions are crucial for TLS studies.In terms of detection,hematoxylin and eosin(H&E),multiplex immunohistochemistry(mIHC),multiplex immunofluorescence(mIF),and 12-chemokine gene signature have been the top approved methods.However,no standard methods exist for the quantitative analysis of TLSs,such as absolute TLS count,analysis of TLS constituent cells,structural features,TLS spatial location,density,and maturity.This study reviews the latest research progress on TLS detection and quantification,proposes new directions for TLS assessment,and addresses issues for the quantitative application of TLSs in the clinic.

Modeling the Cysteine Rich Domain of Plant Metallothionein-like Protein

With the progress of plant genome research, more than 50 plant metallothionein_like (MT_L) genes have been found, but only several MT_L proteins have been detected and no experimental structural information for MT_L proteins has been reported so far. Since detailed knowledge of the protein tertiary structure is required to understand its biological function, a method is needed to determine the structure of these proteins. In this study, the structural data of known mammal MT was used to determine the interatomic distance constraints of the CXC and CXXC motifs and the metal_sulfur chelating cluster. Then several possible MT conformations were predicted using a distance geometry algorithm. The statistical analysis was used to select those with much lower target function values and lower conformation energies as the predicted tertiary structural models of the cysteine_rich (CR) domains of these proteins. A suitable prediction method for modeling the CR domain of the plant MT_L protein was constructed. The accurately predicted result for the known structure of an MT protein from blue crab suggests that this method is practicable. The tertiary structures of CR domains of rape MT_L protein LSC54 was then modeled with this method.

Structure-based insights into recognition and regulation of SAM-sensing riboswitches

Riboswitches are highly conserved RNA elements that located in the 5’-UTR of m RNAs,which undergo real-time structure conformational change to achieve the regulation of downstream gene expression by sensing their cognate ligands.S-adenosylmethionine(SAM)is a ubiquitous methyl donor for transmethylation reactions in all living organisms.SAM riboswitch is one of the most abundant riboswitches that bind to SAM with high affinity and selectivity,serving as regulatory modules in multiple metabolic pathways.To date,seven SAM-specific riboswitch classes that belong to four families,one SAM/SAH riboswitch and one SAH riboswitch have been identified.Each SAM riboswitch family has a well-organized tertiary core scaffold to support their unique ligand-specific binding pocket.In this review,we summarize the current research progress on the distribution,structure,ligand recognition and gene regulation mechanism of these SAM-related riboswitch families,and further discuss their evolutionary prospects and potential applications.

Cloning and bioinformatical analysis of the N-terminus of the sonic hedgehog gene

The sonic hedgehog protein not only plays a key role in early embryonic development, but also has essential effects on the adult nervous system, including neural stem cell proliferation, differentiation migration and neuronal axon guidance. The N-terminal fragment of sonic hedgehog is the key functional element in this process. Therefore, this study aimed to clone and analyze the N-terminal fragment of the sonic hedgehog gene. Total RNA was extracted from the notochord of a Sprague-Dawley rat at embryonic day 9 and the N-terminal fragment of sonic hedgehog was amplified by nested reverse transcription-PCR. The N-terminal fragment of the sonic hedgehog gene was successfully cloned. The secondary and tertiary structures of the N-terminal fragment of the sonic hedgehog protein were predicted using Jpred and Phyre online.

Development of Bioinfo-Portal Tool for the Analysis of Genomic and Proteomic Data

The recent explosion of biological data and the concomitant proliferation of distributed databases make it challenging for biologists and bioinformaticians to discover the best data resources for their needs, and the most efficient way to access and use them For the biologist, running bioinformatics analyses involve a time-consuming management of data and tools. Users need support to organize their work, retrieve parameters and reproduce their analyses. They also need to be able to combine their analytic tools using a safe data flow software mechanism. Finally we have designed a system, Bioinfo-Portal, to provide a flexible and usable web environment for defining and running bioinformatics analyses. It embeds simple yet powerful data management features that allow the user to reproduce analyses and to combine tools using an adobe flex tool. Bioinfo-Portal can also act as a front end to provide a unified view of already-existing collections of bioinformatics resources. Users can analyze genomic and proteomic data by using the tools that has been integrated in the portal （tools for alignments, dotplots, motif detection, domain analysis, profile searching and tertiary structure prediction）. The sequences that user obtained from portal＇s nucleotide and protein databases are easily analyzed by the portal tools on the same interface in no time. User can also take benefit from the animations.

Tertiary lymphoid structures in cancer – considerations for patient prognosis

Tertiary lymphoid structures(TLS)are ectopic lymphoid formations that form within nonlymphoid tissue.They share structural and functional characteristics with secondary lymphoid structures such as lymph nodes and can contain B-cell follicles and germinal centers surrounded by a T-cell region.TLS have been described in several types of cancers and are usually associated with positive patient outcomes.However,TLS differ vastly in cellular composition and location within tissue types.In this review,we discuss factors confounding the interpretation of the evidence for a prognostic role for TLS in cancer and frame these factors in the context of translation to regular clinical use.

Lymphotoxin signalling in tertiary lymphoid structures and immunotherapy

Tertiary lymphoid structures(TLS)often develop at sites of persistent inflammation,including cancers and autoimmune diseases.In most cases,the presence of TLS correlates with active immune responses.Because of their proximity to pathological loci,TLS are an intriguing target for the manipulation of immune responses.For several years,it has become clear that lymphotoxin(LT)signalling plays critical roles in lymphoid tissue organogenesis and maintenance.In the current review,we will discuss the role of LT signalling in the development of TLS.With a focus on cancers and autoimmune diseases,we will highlight the correlations between TLS and disease progression.We will also discuss the current efforts and potential directions for manipulating TLS for immunotherapies.

Tertiary lymphoid structures as unique constructions associated with the organization,education,and function of tumor-infiltrating immunocytes

Tertiary lymphoid structures(TLSs)are formations at sites with persistent inflammatory stimulation,including tumors.These ectopic lymphoid organs mainly consist of chemo-attracting B cells,T cells,and supporting dendritic cells(DCs).Mature TLSs exhibit functional organization for the optimal development and collaboration of adaptive immune response,delivering an augmented effect on the tumor microenvironment(TME).The description of the positive correlation between TLSs and tumor prognosis is reliable only under a certain condition involving the localization and maturation of TLSs.Emerging evidence suggests that underlying mechanisms of the anti-tumor effect of TLSs pave the way for novel immunotherapies.Several approaches have been developed to take advantage of intratumoral TLSs,either by combining it with therapeutic agents or by inducing the neogenesis of TLSs.

Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.

Rice bicoid-related cDNA sequence and its expression during early embryogenesis

Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation. To clone and characterize the rice bicoid-related genes, one cDNA clone, Rb24 (EMBL accession number: AJ2771380), was isolated by screening of rice unmature seed cDNA library. Sequence analysis indicates that Rb24 contains a putative amino acid sequence, which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity, 75% similarity) and involves a lys-9 in putative helix 3. Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner. The transcript was detected strongly in young panicles, but less in young leaves and roots. This results are further confirmed with paraffin section in situ hybridization. The signal is intensive in rice globular embryo and located at the apical tip of the embryo, then, along with the development of embryo, the signal is getting reduced and transfers into both sides of embryo. The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

The expression and antigenicity identification of recombinant rat TGF-β1 in bacteria

In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.

B cells in pancreatic cancer stroma

Pancreatic cancer is a disease with high unmet clinical need.Pancreatic cancer is also characterised by an intense fibrotic stroma,which harbours many immune cells.Studies in both human and animal models have demonstrated that the immune system plays a crucial role in modulating tumour onset and progression.In human pancreatic ductal adenocarcinoma,high B-cell infiltration correlates with better patient survival.Hence,B cells have received recent interest in pancreatic cancer as potential therapeutic targets.However,the data on the role of B cells in murine models is unclear as it is dependent on the pancreatic cancer model used to study.Nevertheless,it appears that B cells do organise along with other immune cells such as a network of follicular dendritic cells(DCs),surrounded by T cells and DCs to form tertiary lymphoid structures(TLS).TLS are increasingly recognised as sites for antigen presentation,T-cell activation,Bcell maturation and differentiation in plasma cells.In this review we dissect the role of B cells and provide directions for future studies to harness the role of B cells in treatment of human pancreatic cancer.

Germinal center-dependent and-independent immune responses of tumor-infiltrating B cells in human cancers

B cells play essential roles in immunity,mainly through the production of high affinity plasma cells(PCs)and memory B(Bmem)cells.The affinity maturation and differentiation of B cells rely on the integration of B-cell receptor(BCR)intrinsic and extrinsic signals provided by antigen binding and the microenvironment,respectively.In recent years,tumor infiltrating B(TIL-B)cells and PCs(TIL-PCs)have been revealed as important players in antitumor responses in human cancers,but their interplay and dynamics remain largely unknown.In lymphoid organs,B-cell responses involve both germinal center(GC)-dependent and GC-independent pathways for Bmem cell and PC production.Affinity maturation of BCR repertoires occurs in GC reactions with specific spatiotemporal dynamics of signal integration by B cells.In general,the reactivation of high-affinity Bmem cells by antigens triggers GC-independent production of large numbers of PC without BCR rediversification.Understanding B-cell dynamics in immune responses requires the integration of multiple tools and readouts such as single-cell phenotyping and RNA-seq,in situ analyses,BCR repertoire analysis,BCR specificity and affinity assays,and functional tests.Here,we review how those tools have recently been applied to study TIL-B cells and TIL-PC in different types of solid tumors.We assessed the published evidence for different models of TIL-B-cell dynamics involving GC-dependent or GC-independent local responses and the resulting production of antigen-specific PCs.Altogether,we highlight the need for more integrative B-cell immunology studies to rationally investigate TIL-B cells as a leverage for antitumor therapies.

Dendritic cell maturation in the tumor microenvironment

Maturation is essential for dendritic cells(DCs)to function as a bridge between innate and adaptive immunity.The plastic maturation states correspond to the multifaceted roles of DCs in the initiation of protective,tolerogenic and pathogenic immune responses.The formation of a distinct maturation state of DCs involves the complex crosstalk between the intrinsic differences in their ontogeny and the immune environments.Recent studies have defined a universal mature DC population in the tumor microenvironment(TME),but their ontogenesis and functions remain unclear.Here,we summarize recent advances in DC maturation and attempt to dissect the complex functions of mature DCs in the TME.We highlighted that there is a distinct similarity and heterogeneity of mature DCs in the TME,in which the environment and development are both important.These help us revisit the diversity of DC maturation and to better dissect the complex functions of DCs in the TME.

Structural diversity of eukaryotic 18S rRNA and its impact on alignment and phylogenetic reconstruction

Ribosomal RNAs are important because they catalyze the synthesis of peptides and proteins.Comparative studies of the secondary structure of 18S rRNA have revealed the basic locations of its many length-conserved and lengthvariable regions.In recent years,many more sequences of 18S rDNA with unusual lengths have been documented in GenBank.These data make it possible to recognize the diversity of the secondary and tertiary structures of 18S rRNAs and to identify the length-conserved parts of 18S rDNAs.The longest 18S rDNA sequences of almost every known eukaryotic phylum were included in this study.We illustrated the bioinformatics-based structure to show that,the regions that are more length-variable,regions that are less length-variable,the splicing sites for introns,and the sites of A-minor interactions are mostly distributed in different parts of the 18S rRNA.Additionally,this study revealed that some length-variable regions or insertion positions could be quite close to the functional part of the 18S rRNA of Foraminifera organisms.The tertiary structure as well as the secondary structure of 18S rRNA can be more diverse than what was previously supposed.Besides revealing how this interesting gene evolves,it can help to remove ambiguity from the alignment of eukaryotic 18S rDNAs and to improve the performance of 18S rDNA in phylogenetic reconstruction.Six nucleotides shared by Archaea and Eukaryota but rarely by Bacteria are also reported here for the first time,which might further support the supposed origin of eukaryote from archaeans.

A Novel Structural Measure Separating Non-Coding RNAs from Genomic Backgrounds

RNA secondary structure has become the most exploitable feature for ab initio detection of non-coding RNA（nc RNA） genes from genome sequences. Previous work has used Minimum Free Energy（MFE） based methods developed to identify nc RNAs by measuring sequence fold stability and certainty. However, these methods yielded variable performances across different nc RNA species. Designing novel reliable structural measures will help to develop effective nc RNA gene finding tools. This paper introduces a new RNA structural measure based on a novel RNA secondary structure ensemble constrained by characteristics of native RNA tertiary structures. The new method makes it possible to achieve a performance leap from the previous structure-based methods. Test results on standard nc RNA datasets（benchmarks） demonstrate that this method can effectively separate most nc RNAs families from genome backgrounds.