Personalized treatment of perihilar cholangiocarcinoma based on tumor genetic and molecular characteristics

This editorial discusses the article written by Tchilikidi et al that was published in the latest edition of the World Journal of Gastrointestinal Surgery.Genetic and molecular profiling of perihilar cholangiocarcinoma(pCCA)has identified a number of key abnormalities that drive tumor growth and spread,including pyruvate kinase M2,proline rich 11,and transcription factor 7,etc.pCCA has specific genetic and molecular features that can be used to develop personalized treatment plans.Personalized treatment approaches offer new opportunities for effectively targeting the underlying drivers of tumor growth and progression.The findings based on tumor genetic and molecular characteristics highlight the importance of developing personalized treatment strategies.

Molecular characteristics and structure–activity relationships of food-derived bioactive peptides

Peptides are functional active fragments of proteins which can provide nutrients needed for human growth and development,and they also have unique physiological activity characteristics relative to proteins.Bioactive peptides contain a great deal of development potential.More specifically,food-derived bioactive peptides have the advantages of a wide variety of sources,unique structures,high efficiency and safety,so they have broad development prospects.This review provides an overview of the current advances regarding the preparation,functional characteristics,and structure–activity relationships of food-derived bioactive peptides.Moreover,the prospects for the future development and application of food-derived bioactive peptides are discussed.This review may provide a better understanding of foodderived bioactive peptides,and some constructive inspirations for further research and applications in the food industry.

Molecular Characteristics and Phylogenetic Analysis of N Gene of Human Derived Rabies Virus

Objective To investigate the relationship between the molecular characteristics and phylogenetic evolution of rabies N gene.Methods Saliva samples were collected from rabies cases,and RT-PCR was used to amplify the N gene of rabies virus with the specific primers.The amplifying product of RT-PCR was cloned to pUCm-T vector and transformed into E.coli XL1-Blue and then the blue-white selection,PCR screening and gene sequencing were carried out to identify the positive clones.Finally,ExPASy and other bioinformatics softwares were used to analyze and predict the structure and biological characteristics of the N genome.Results The amplification product of RT-PCR was 1 353 bp,the recombinant plasmid pUCm-T/N was constructed,the whole length of the N gene open reading frame was composed of 1 353 nucleotide residues to code 450 amino acids （20 kinds）,the accession number submitted to the Genbank was HM756692,its sequence homology of nucleotides and amino acids compared with the vaccine strain CTN-1-V was 90% and 99% respectively.The evolutionary analysis showed that the isolated strain belonged to genotype I with certain geographic regionality.Conclusion The characteristics investigation and bioinformatics analysis of Hunan0806 N gene will provide fundament data to reveal the significance of the N gene characteristics for rabies epidemiology and its prevention control.

Molecular Characteristics of New Wheat Starch and Its Digestion Behaviours

In order to understand the effect of starch molecular characteristics on the gel structure, which subsequently influence the gel digestion behaviours, three wheat starches, control （conventional wheat starch）, two new wheat cultivars with different genetic backgrounds （by knocking out SBE IIb and SBE IIa, respectively） were used in this study. In comparison with control, slight differences in the morphology of the starch granules of new wheat 1 were observed, whereas the starch granules of new wheat 2 had irregular shapes both for A-type granules and B-type granules. Starch molecular weight size was determined by SE-HPLC, and the results indicate that there was a subtle increase in the amylose content in the starch of new wheat 1 compared to that of control. The starch of new wheat 2 had the highest amylose content, and the molecular weight （MW） of its amylopectin was the lowest among the three starches. Fourier transform infrared spectroscopy （FTIR） was employed to investigate starch gel structure and the results suggest that the molecules of starch gel from new wheat 2 are more likely to re-associate to form an organized conformation. The digestion behaviours of the three starch gels were measured using a mixture of pancreatin a-amylase and amyloglucosidase. The results indicated that the starch gels of control and new wheat 1 had very high digestibility of 91.7 and 91.9%, respectively, whereas the digestibility of wheat 2 starch gel was only 36.2%. In comparison with the digestion curve pattems of control and new wheat 1 starch gels, the new wheat 2 exhibited a much lower initial velocity. These results indicated that the molecules in the starch of new wheat 2 are more readily to re-associate to form an organized structure during gel formation because of its unique molecular characteristics.

Molecular Characteristics of S1 Gene of Infectious Bronchitis Virus Isolated from Chicken Proventriculus

Infectious bronchitis virus was isolated from swollen proventriculi of clinically ill chicken. The suspected virus samples (2/97, 3/97, 1/98) were adapted in SPF chicken embryos for virus isolation and identification. All the virus isolates were able to agglutinate chicken erythrocytes after treatment with trypsin, and interfer with the reproduction of Newcastle disease virus in chicken embryos, and have low antigenic relat-edness values with reference positive IBV. The isolates 2/97, 3/97, 1/98 RNAs extracted from the allantoic fluid of inoculated embryonated eggs were converted to cDNA by reverse transcription with 3'-primer of S1 gene of (IBV). Polymerase chain reaction (PCR) was performed with two primers which span the S1 gene. Amplified product of 1. 93 kb was subjected to EcoR I and BamH I digestion and the fragments obtained were the same as expected size. The PCR product was ligated to pBlueScript-SK ( + ) vector, and its nucleotide sequence was determined by the dideoxy-mediated chain termination method. Nucleotide sequence analysis showed 73. 6 - 99. 7% homology between the isolated IBV and the IBV strains in GenBank. The homology of amino acid was 71. 4 - 99.4%.

Molecular characteristics and antimicrobial susceptibility of Staphylococcus aureus among children with respiratory tract infections in southwest China

Background The molecular characteristics and antimicrobial susceptibility of Staphylococcus aureus (S.aureus) in general pediatric wards and county-level hospitals were rarely reported in China.Methods Staphylococcus aureus was isolated from children hospitalized with respiratory tract infection (RTI) in Zhongjiang and Youyang counties in 2015.All isolates were typed by multilocus sequence,staphylococcal protein A,accessory gene regulator (agr),and staphylococcal cassette chromosome mec [SCCmec,for methicillin-resistant S.aureus (MRSA) only].Polymerase chain reaction was used to screen 21 super-antigen (SAg) genes and panton-valentine leukocidin (pvl).Antimicrobial susceptibility testing was performed by E test.Results A total of 2136 children were enrolled.Overall,125 (5.9%) children carried S.aureus,among which MRSA accounted for 42.4%.ST59-SCCmec type Ⅳ-t437-agr group Ⅰ (58.5%) was the most prevalent genotype in MRSA,and ST188-t189-agr group Ⅰ (22.2%) was the top genotype in methicillin-sensitive S.aureus (MSSA).The pvl carriage rate in MRSA and MSSA was 15.1% and 9.7%,respectively (P =0.4112).About 96.8% of S.aureus isolates were positive for at least one SAg gene.The most common SAg gene profile in the dominant ST59 clone was seb-sek-seq (42.8%).All S.aureus isolates were resistant to penicillin and erythromycin (minimum inhibitory concentration 90 was > 32 and 256 mg/L to penicillin and erythromycin,respectively),but usually susceptible to other tested non-β-lactam antimicrobials.Conclusions Staphylococcus aureus and MRSA were detected with a high frequency in children with RTI in county-level hospitals of China.ST59-SCCmec type Ⅳ-t437-agr group Ⅰ was the dominant MRSA clone.The S.aureus isolates exhibited high resistance to penicillin and erythromycin.

Molecular genetics of early-onset colorectal cancer

Early-onset colorectal cancer(EOCRC)has been rising in global prevalence and incidence over the past several decades.Environmental influences,including generational lifestyle changes and rising obesity,contribute to these increased rates.While the rise in EOCRC is best documented in western countries,it is seen throughout the world,although EOCRC may have distinct genetic mutations in patients of different ethnic backgrounds.Pathological and molecular characterizations show that EOCRC has a distinct presentation compared with later-onset colorectal cancer(LOCRC).Recent studies have identified DNA,RNA,and protein-level alterations unique to EOCRC,revealing much-needed biomarkers and potential novel therapeutic targets.Many molecular EOCRC studies have been performed with Caucasian and Asian EOCRC cohorts,however,studies of other ethnic backgrounds are limited.In addition,certain molecular characterizations that have been conducted for LOCRC have not yet been repeated in EOCRC,including high-throughput analyses of histone modifications,mRNA splicing,and proteomics on large cohorts.We propose that the complex relationship between cancer and aging should be considered when studying the molecular underpinnings of EOCRC.In this review,we summarize current EOCRC literature,focusing on sporadic molecular alterations in tumors,and their clinical implications.We conclude by discussing current challenges and future directions of EOCRC research efforts.

Molecular Biomarker Characteristics of the Linxi Formation Source Rocks in the Middle-Western Region of Inner Mongolia:New evidence for late-stage tectonic evolution of the Paleo-Asian Ocean

Objective Two important geological issues have long been controversial in the Xing-Meng area of North China. The first concerns the final closure of Paleo-Asian Ocean in Xing-Meng area, and the other concerns the folding and lifting of the Xing-Meng Trough. The focus of thses issues is the Late Permian sedimentary environment, which is generally considered to be either an exclusively continental environment or from the closed inland sea environment in the Early to Middle stage to continental lacustrine environment in the late stage. In recent years, we have successively discovered abundant typical marine fossils （e.g., bryozoans and calcareous algae） in the Upper Permian thick limestone layer from Linxi County and Ar Horqin Banner in eastern region of Inner Mongolia and Jiutain County in Jilin Province. These significant findings have attracted the attention from fellow academics.

Analytical characteristics of a qPCR-based molecular diagnostic assay-conceptual considerations for laboratory personnel

Dear Editor：Quantitative real-time PCR has revolutionized molecular diagnostics with its ease of use,increased sensitivity and specificity and low turnaround time.PCR/quantitative PCR（qPCR）-based assays offer a distinct advantage over other serological/conventional diagnostic approaches.The ability to diagnose infectious diseases has benefited from the availability of US FDA approved and Conformite Europeenne（CE）-marked qPCR-based in-vitro diagnostic kits from international companies.The high-quality kits are calibrated with the World Health Organization（WHO）reference standards and the National Institute for Biological Standards and Control（NIBSC）standards.

Three anti-inflammatory polysaccharides from Lonicera japonica Thunb.:insights into the structure-function relationships

This study demonstrates the feasibility of producing three polysaccharides(neutral LJP-1,acidic LJP-2 and acidic LJP-3)with significant in vitro and in vivo anti-inflammatory activities from the flowers of Lonicera japonica.The three polysaccharides differed in chemical composition,molecular weight(Mw)distribution,glycosidic linkage pattern,functional groups and morphology.They exhibited excellent protective effects(in a dose-dependent manner)in lipopolysaccharide-injured RAW264.7 macrophages and Cu SO4-damaged zebrafish via reducing NO production and inhibiting the overexpressions of inflammation-related transcription factors,inflammatory proteins and cytokines in the NF-κB/MAPK signaling pathways.Their antiinflammatory effects varied owing to their different molecular characteristics and chemical compositions.Overall,LJP-2 at 400μg/m L was the most effective.LJP-2 consisted mainly of→5)-α-L-Araf(1→,→4)-α-LGalp A(1→and→2)-α-L-Rhap(1→residues with terminal T-β-D-Glcp.Thus,honeysuckle flowers are good sources of anti-inflammatory polysaccharides,and precise fractionation enables the production of potent antiinflammatory agents for the development of functional foods and healthcare products.

Molecular Characterization of Viral G Gene in Emerging and Re-emerging Areas of Rabies in China, 2007 to 2011

In recent years (2007 to 2011), although the overall number of rabies cases in China has decreased, there is evidence of emerging or re-emerging cases in regions without previous rabies cases or with low incidence of rabies. To investigate the origin and the factors affecting the spread of rabies in China, specimens were collected from 2007 to 2011 from provinces with emerging and re-emerging cases and tested for the presence of the rabies virus. Positive specimens were combined with sequences from GenBank to perform comparisons of homology and functional sites, and to carry out phylogenetic analyses. Out of these regions, five provinces had 9 positive specimens from canine and cattle, and 34 canine or human specimens were obtained from previously high-incidence provinces. Complete sequences of G gene were obtained for these samples. Homology of the sequences of these 43 specimens was 87%-100% at the nucleotide level and 93.7% -100% at the amino acid level. These G gene sequences were combined with reference sequence from GenBank and used to construct a phylogenetic tree. The results showed that 43 specimens were all assigned to China clade I and clade II, with all specimens from emerging and re-emerging areas placed within clade I. Specimens isolated from Shanxi and Inner Mongolia in 2011 were distinct from previously-isolated local strains and had closer homology to strains from Hebei, Beijing and Tianjin whereas new isolates from Shanghai were tightly clustered with strains isolated in the 1990s. Finally, Shaanxi isolates were clustered with strains from adjacent Sichuan. Our results suggest that the rabies cases in emerging and re-emerging areas in China in the last 5 years are a consequence of the epidemic spreading from of neighboring provinces and regions experiencing a serious epidemic of rabies.

Identification and Biological Characteristics of Blank Spot Pathogen from Lilium lancifolium

With the increasing planting area of Lilium lancifolium, the leaf fungal dis-ease of L. lancifolium is becoming more and more serious. In June and July of 2014, the excessive rainfal leads to the serious outbreak of leaf disease of L. lanci-folium. In mid-June, the wilting rate of L. lancifolium in seriously-infected field was even up to 50%-70%. In some fields, the shoots of L. lancifolium even al wilted. The pathogen was isolated from the infected leaves of L. lancifolium. Its pathogenici-ty, spore morphology, 18S rDNA sequence and biological characteristics were stud-ied. The results showed the isolated pathogen was Alternaria alternate. The lethal temperature of mycelial growth was 55 ℃. The optimum pH was 6-7. Among the tested carbon sources and nitrogen sources, the optimum carbon source was mal-tose, and the optimum nitrogen source was yeast extract.

Molecular Epidemiology of Klebsiella pneumoniae from Clinical Bovine Mastitis in Northern Area of China,2018–2019

Klebsiella pneumoniae(K.pneumonia,KpⅠ)is a predominate inducement of bovine mastitis,which is associated with high mortality and milk yield reduction.However,data is lacking on the molecular characteristics of bovine K.pneumoniae,limiting the risk assessment of its transmission through the food chain.Herein,we investigated the prevalence of K.pneumoniae in 6301 clinical mastitis(CM)milk samples from dairy cattle in northern area of China.In total,183 K.pneumoniae isolates were recovered,with detection rates of 3.0% and 2.8% in 2018 and 2019,respectively.Like human clinical K.pneumoniae,all CM K.pneumoniae isolates belonged to one of three phylogroups:KpⅠ(n=143),Klebsiella.quasipneumoniae subsp.similipneumoniae(KpⅡ-B)(n=37),and Klebsiella variicola(KpⅢ)(n=3).We detected the extendedspectrum β-lactamase-encoding genes bla_(SHV-2a),blac_(CTX-M-14),and bla_(CTX-M-15),as well as clpC,lpfA,lacI,lacZ,lacY,and the fecABDEIR operon in the KpⅠ isolates,which may contribute to their pathogenicity and host adaptability in cows.The high prevalence of KpⅠ in dairy farms may be problematic,as it showed relatively higher rates of antibiotic resistance and virulence gene carriage than the KpⅡ-B and KpⅢ isolates.Furthermore,we observed distinct differences in population structure between CM-and human infection-associated KpⅠ isolates,with the genes associated with invasive infection in humans rarely being observed in bovine isolates,indicating that few CM-associated K.pneumoniae isolates pose a threat to human health.Nevertheless,bovine KpⅡ-B isolates shared a high level of nucleotide sequence identity with isolates from human infections and frequently carried the nitrogen-fixation gene nif,suggesting an association between KpⅡ-B isolates from cattle and humans,and plant-derived bacteria.

Cloning and Bioinformatics Analysis of Donkey Growth Hormone Gene cDNA

[Objective] The paper aimed to study the donkey GH gene features and functions.[Method] A pair of specific PCR primers was designed for cloning the coding sequence of the donkey GH gene from liver tissue.[Result] 706 bp fragment was got by RT-PCR amplification.The sequence included a complete open reading frame and encoded 216 amino acids.The protein encoded by donkey GH gene had seven hydrophobic region,seven transmembrane regions and a signal peptide;it＇s secondary structure had α-helix and irregular curl and three-dimensional solution structure composed of 27-215 amino acids.[Conclusion] GH gene of donkey was very conservative in evolution.The phylogenetic tree constructed basing on CDS sequence is consistent with the results of comparative morphology and comparative physiology.

Characterization of Protein in Old Myocardial Infarction by FTIR Micro-spectroscopy

The aim of the present study was to assess whether Fourier transform infrared spectrometry (FTIR) micro-spectroscopy could produce distinct spectral information on protein of old myocardial infarction (OMI) and to set them as molecular markers to diagnose atypical OMI. Paraffin-embedded heart samples were derived from victims dying of OMI. In combination with histological stain, FTIR and infrared micro-spectroscopy, the characteristics of OMI were analyzed morphologically and molecularly. The most relevant bands identified were the amide A, B, Ⅰ and Ⅱ, showing crucial spectral differences between apparent normal region and OMI region, including the peak position blue shift and the increased intensity of OMI, moreover relative increase in α-helix and decrease in β-sheet of protein secondary structures in OMI. Comparing to single spectral band, the I1650/I1550 ratio was increased and rationally used as a molecular marker for diagnosing OMI. These novel preliminary findings supported further exploration of FTIR molecular profiling in clinical or forensic study, and were in accordance with histopathology.

Research on human glioma stem cells in China

Research on human glioma stem cells began early in the 21st century and since then has become a rapidly growing research field with the number of publications increasing year by year. The research conducted by our diverse group of investigators focused primarily on cell culture techniques, molecular regulation, signaling pathways, cancer treatment, the stem cell microenvironment and the cellular origin and function of glioma stem cells. In particular, we put forward our view that there are inverse or forward transformations among neural stem cells, glial cells and glioma stem cells in glioma tissues under certain conditions. Based on the background of the progress of international research on human glioma stem cells, we aim to share our progress and current findings of human glioma stem cell research in China with colleagues around the world.

Biochar-derived dissolved organic matters influencing bacterium characteristics during biodegradation of sulfamethoxazole and chloramphenicol under alternation of visible and avoiding light

The influence of biochar-released dissolved organic matter(BDOM)on the transcription of gene(DEG)in Pseudomonas stutzeri and Shewanella putrefacien during sulfamethoxazole(SMX)and chloramphenicol(CAP)biodegradation under visible light was investigated in this study.The results indicated that BDOM components would be nutrients for bacterial amplification and growth under the culture conditions of xenon lamp irradiation and avoiding light,especially BDOM from low temperatures.Additionally,visible light irradiation would improve the saturated fatty acid by stimulating the cell membrane of the microorganism,thus promoting the biodegradation of antibiotics through altering P.stutzeri and S.putrefaciens reoxidative and catabolism processes and significantly inhabiting the copy number of their genes.Moreover,the upregulated genes and enzymes related to SMX and CAP-metabolic and catabolic processes were enriched,which were involved in the pathways of biodegradation,further improving biodegradation efficiency.In particular,interaction network analysis between the top 100 dominant functional genes from P.stutzeri and S.putrefaciens and the molecular types of BDOM,e.g.,CHO,CHON,and CHOS(p<0.05),indicated that the genes of molecular function showed a high positive or negative correlation with the CHO type of BDOM.The results revealed that the CHO type of BDOM affected the functional genes of molecular function,cellular component,and biological process from P.stutzeri and S.putrefaciens,influencing the biodegradation of SMX and CAP.This study provided an basis for BDOM playing a role in antibiotic removal from the aqueous solution using biochar combined with photobiodegradation.

Antimicrobial resistance and molecular epidemiological characteristics of clinical isolates of Staphylococcus aureus in Changsha area

Background Increasing prevalence of Staphylococcus aureus （S. aureus）, particularly methicillin-resistant S. aureus （MRSA） has been reported in China. In this study, we investigated the drug resistance characteristic, genetic background, and molecular epidemiological characteristic of S. aureus in Changsha. Methods Between January 2006 and December 2008, 293 clinical isolates of S. aureus were collected from 11 hospitals in Changsha and identified by the Vitek-2 system. All the isolates were verified as MRSA by PCR amplification of both femA and mecA genes. K-B disk method was used to test drug sensitivity of S. aureus to antibiotics. Pulsed-field gel electrophoresis （PFGE） was performed for genotypic and homologous analysis of 115 isolates randomly selected from the original 293 clinical S. aureus isolates. Results S. aureus was highly resistant to penicillin, ampicillin, erythromycin, and clindamycin with resistant rates of 96.6%, 96.6%, 77.1%, and 67.2% respectively. All the isolates were susceptible to tecoplanin, vancomycin, and linezolid. MRSA accounted for 64.8% （190/293） of all the S. aureus strains. The 115 S. aureus isolates were clustered into 39 PFGE types by PFGE typing, with 13 predominant patterns （designated types A to M） accounting for 89 isolates. The most prevalent PFGE type was type A （n=56, 48.7%） and 100.0% of type A strains were MRSA. PFGE type A included 13 subtypes, and the most prevalent subtype was subtype A1 （46.4%, 26/56）. Strains with PFGE type A were isolated from eight hospitals （8/11）, and both subtypes A1 and A4 strains were isolated in a university hospital. Conclusions Clinical isolates of S. aureus in Changsha were resistant to multiple traditional antibiotics. There was an outbreak of PFGE type A MRSA in this area and the A1 subtype was the predominant epidemic clone. Dissemination of the same clone was an important reason for the wide spread of MRSA.

Proteomic signatures of infiltrative gastric cancer by proteomic and bioinformatic analysis

BACKGROUND Proteomic signatures of Ming's infiltrative gastric cancer(IGC)remain unknown.AIM To elucidate the molecular characteristics of IGC at the proteomics level.METHODS Twelve pairs of IGC and adjacent normal tissues were collected and their proteomes were analyzed by high performance liquid chromatography tandem mass spectrometry.The identified peptides were sequenced de novo and matched against the SwissProt database using Maxquant software.The differentially expressed proteins(DEPs)were screened using|log2(Fold change)|>1 and P-adj<0.01 as the thresholds.The expression levels of selected proteins were verified by Western blotting.The interaction network of the DEPs was constructed with the STRING database and visualized using Cytoscape with cytoHubba software.The DEPs were functionally annotated using clusterProfiler,STRING and DAVID for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways.P<0.05 was considered statistically significant.RESULTS A total of 7361 DEPs were identified,of which 94 were significantly up-regulated and 223 were significantly down-regulated in IGC relative to normal gastric tissues.The top 10 up-regulated proteins were MRTO4,BOP1,PES1,WDR12,BRIX1,NOP2,POLR1C,NOC2L,MYBBP1A and TSR1,and the top 10 down-regulated proteins were NDUFS8,NDUFS6,NDUFA8,NDUFA5,NDUFC2,NDUFB8,NDUFB5,NDUFB9,UQCRC2 and UQCRC1.The up-regulated proteins were enriched for 9 biological processes including DNA replication,ribosome biogenesis and initiation of DNA replication,and the cellular component MCM complex.Among the down-regulated proteins,17 biological processes were enriched,including glucose metabolism,pyruvic acid metabolism and fatty acidβ-oxidation.In addition,the mitochondrial inner membrane,mitochondrial matrix and mitochondrial proton transport ATP synthase complex were among the 6 enriched cellular components,and 11 molecular functions including reduced nicotinamide adenine dinucleotide dehydrogenase activity,acyl-CoA dehydrogenase activity and nicotinamide adenine dinucleotide binding were also enriched.The significant KEGG pathways for the up-regulated proteins were DNA replication,cell cycle and mismatch repair,whereas 18 pathways including oxidative phosphorylation,fatty acid degradation and phenylalanine metabolism were significantly enriched among the down-regulated proteins.CONCLUSION The proteins involved in cell cycle regulation,DNA replication and mismatch repair,and metabolism were significantly altered in IGC,and the proteomic profile may enable the discovery of novel biomarkers.

Effects of Electron Donating Ability of Substituents and Molecular Conjugation on the Electronic Structures of Organic Radicals

The geometries and electronic structures of a series of electron donor-acceptor radical molecules have been studied theoretically.The computational results show that the introduction of substituents with strong electron donating ability into tri-(2,4,6-trichlorophenyl)methyl(TTM)radicals enables the radical molecules to form the non-Aufbau electronic structure.The difficulty of forming the non-Aufbau electronic structure decreases with the enhancement of the electron donating ability of the substituent,but the expansion of the molecular conjugated system is not conducive to the formation.The hybridization of different fragments in molecular orbitals results in the disproportionation of orbital energy level and forms a staggered energy level structure.The electronic structure of radical molecules can be adjusted by substituents and molecular skeleton profoundly,which is a very effective means for molecular design.