As a result, nine strains from humans, animals, and foods were positive for tmexCD1-toprJ1, which was located on plasmids or the chromosome. Seven different sequence types (STs) were identified, including ST15 (n = 2), ST580, ST1944, ST2294, ST5982, ST6262 (n = 2), and ST6265. All the positive strains were clustered into two distinct clades, and they shared a 24,087-bp core structure of tmexCD1-toprJ1, bounded by IS26 in the same orientation. IS26 could facilitate rapid and wide dissemination of tmexCD1-toprJ1 in Enterobacteriaceae from various sources.In this study, we screened the IMPORTANCE Tigecycline has been regarded as one of the last-resort antibiotics available for the treatment of infections caused by carbapenem-resistant Enterobacterales. The plasmid-mediated resistance-nodulation-division–type efflux pump gene cluster tmexCD-toprJ is a newly identified tigecycline resistance determinant. In this study, we revealed that tmexCD-toprJ has disseminated among Klebsiella pneumoniae strains from poultry, food markets, and patients. It is critical to strengthen continuous monitoring, and control measures should be implemented to prevent the further dissemination of tmexCD-toprJ. Enterobacterales (CRE) is an urgent and growing threat to the global public health  containing tigecycline (2 mg/liter). Species identification was performed using matrix-assisted laser desorption ionization–time-of-flight mass spectrometry . The isolates were further screened for the tmexCD-toprJ gene cluster by PCR and Sanger sequencing as previously described (n = 1), animals (n = 6), and foods (n = 2) were positive for tmexCD1-toprJ1 (Table S1). Antibiotic susceptibility testing was performed using the agar dilution method or broth microdilution method (limited to colistin and tigecycline). The results were interpreted according to the 2020 Clinical and Laboratory Standards Institute guidelines (document M100-S30), except the breakpoints of streptomycin, tigecycline, and florfenicol were interpreted according to European Committee on Antimicrobial Susceptibility Testing guidelines (https://www.eucast.org/). Nine tmexCD1-toprJ1-positive strains exhibited resistance to tigecycline  and were also resistant to ampicillin, cefazolin, gentamicin, amikacin, streptomycin, tetracycline, chloramphenicol, florfenicol, nalidixic acid, ciprofloxacin, and sulfamethoxazole-trimethoprim. Resistance to colistin  was observed in YZ22PK089 .A total of 35 pig fecal samples were collected from 3 farms; 206 pet fecal samples were collected from 3 pet hospitals; 235 meat samples, including 117 pork and 118 chicken meat, were obtained from 2 local farmer markets; and 158 chicken intestinal content samples were collected from 4 slaughterhouses. In addition, 309 human feces samples were acquired from healthy donors. All the samples were gathered from June 2021 to September 2022 in Yangzhou, Jiangsu Province, China . Simultaneously, 598 nonrepetitive clinical Z22PK089 . Only onPRJNA893449). The de novo hybrid assembly was performed using SPAdes 3.11 (In silico multilocus sequence typing (MLST) was performed in the Institute Pasteur MLST online database (https://bigsdb.pasteur.fr/klebsiella/). A total of seven STs were identified, namely, ST15 (n = 2), ST580, ST1944, ST2294, ST5982, ST6262 (n = 2), and ST6265, among which ST6262 and ST6265 were novel STs (http://www.genomicepidemiology.org/). In addition to tmexCD1-toprJ1, all nine strains harbored blaDHA-1, aph(4)-Ia, aac(3)-IV, aadA1, aadA2, strA/B, armA, oqxAB, qnrB4, cmlA1, fosA, sul1, sul3, mph(E), and msr(E). Resistance gene mcr-1 was identified in YZ22PK089 (http://chiplot.online/#) was used to visualize the phylogenetic tree. The nine tmexCD-toprJ-positive K. pneumoniae isolates were divided into two clades (Whole-genome sequencing was subsequently performed using Illumina sequencing (NovaSeq) and Oxford Nanopore (MinION system) platforms  and the chromosome (n = 1) (Table S4). After comparing the sequences of eight plasmids carrying tmexCD1-toprJ1 , it was found that only the plasmid pYZ22CK024_1 contained the complete conjugative transfer region, whereas the other plasmids were missing the desired region  or had an incomplete conjugation transfer-related gene TraG (pSBH193_1 and pYZ22CS023_1). This may explain why only one strain, YZ22CK024, was able to transfer tigecycline resistance to E. coli C600 through conjugation. All the tmexCD1-toprJ1-carrying plasmids or chromosome shared a 24,087-bp conserved core structure [IS26-aph(3′)-Ia-IS26-Δhp2-tnfxB1-tmexCD1-toprJ1-ΔtnpA-tnpR-strA/B-ΔIS903-ΔTn1721-IS26-virB10-hp-ΔTn5393-ISEc59-aph(4)-Ia-aac(3)-Iv-IS26], which was identical to that in K. pneumoniae plasmids p18-29-MDR (GenBank accession number MK262712) and pHN111RT-1 (GenBank accession number MT647838) , except that the IS26-aph(3′)-Ia fragment was absent. The 21,381-bp segment [IS26-Δhp2-tnfxB1-tmexCD1-toprJ1-ΔtnpA-tnpR-strA/B-ΔIS903-ΔTn1721-IS26-virB10-hp-ΔTn5393-ISEc59-aph(4)-Ia-aac(3)-Iv-IS26] in pHN111RT-1 and pHN111WT-1 was previously confirmed to form a circular intermediate  . The corrmediate , suggesttmexCD1-toprJ1 has disseminated among K. pneumoniae strains from poultry, food markets, and patients. IS26-mediated horizontal transfer of tmexCD1-toprJ1 could facilitate its rapid and wide dissemination in Enterobacteriaceae from various sources. It is critical to strengthen continuous monitoring, and control measures should be implemented to prevent the further dissemination of tmexCD-toprJ.In conclusion, our results showed that This study was approved by Jiangsu Key Laboratory of Zoonosis and Clinical Medical College, Yangzhou University, Yangzhou, China.PRJNA893449.The genome sequences in this study have been deposited into NCBI GenBank under BioProject accession number


COVID-19 has indirectly led to excess deaths attributable to other illnesses. Pre-pandemic studies in India have shown mortality rates of 3-12% amongst young children hospitalized with suspected pneumonia. Here, we describe mortality among young children hospitalized with pneumonia during the COVID-19 pandemic.Children aged 1-35 months hospitalized with suspected pneumonia were enrolled prospectively across six sites in India. Demographic and clinical information were obtained, and children were followed until 90 days after hospital discharge.Between October 2020 and November 2022, 4,517 children with suspected pneumonia were enrolled and 4,443 (98.4%) with known mortality status were included in this analysis. Monthly average enrollment was 104 before and during the SARS-CoV-2 peak Delta period (April–June 2021) and 213 post-peak-Delta period (July 2021–November 2022). 71% had WHO-defined severe pneumonia, 26% had SpO2< 90% at admission, 39 of 3000 tested were SARS-CoV-2 positive, and 17% left against medical advice (LAMA). Of 723 total deaths =16%), 42% were detected in-hospital, 23% occurred within 48 hours after discharge/LAMA, and 35% occurred 2-90 days after discharge/LAMA. CFR was higher among children before and during than post Delta peak  but average number of deaths per month was similar (28.2 vs. 27.6). Mortality was higher in LAMA cases  and accounted for most (57%) post-hospitalization deaths.A high CFR (16%) was observed for young children hospitalized for pneumonia in India, with 58% of deaths occurring after discharge. Deaths were higher during the-Delta and Delta wave wave of the COVID-19 pandemic. Although very few children had COVID-19, pandemic-related restrictions and late presentation to healthcare may have contributed to high mortality from pneumonia.All Authors: No reported disclosures


Pseudomonas syringae pv. tomato (Pst) DC3000 functions to overcome plant immunity. We previously isolated eight Arabidopsis (scord) mutants that exhibit increased susceptibility to a COR-deficient mutant of Pst DC3000. Among them, the scord6 mutant exhibits defects both in stomatal closure response and in restricting bacterial multiplication inside the apoplast. However, the identity of SCORD6 remained elusive.Plants mount coordinated immune responses to defend against pathogens. However, the cellular components required for plant immunity are not fully understood. The jasmonate-mimicking coronatine (COR) toxin produced by SCORD6 gene.In this study, we aim to identify the SCORD6 via next-generation sequencing and found it to be MURUS1 (MUR1), which is involved in the biosynthesis of GDP-L-fucose.We identified SCORD6 as MUR1 led to a series of experiments that revealed a multi-faceted role of L-fucose biosynthesis in stomatal and apoplastic defenses as well as in pattern-triggered immunity and effector-triggered immunity, including glycosylation of pattern-recognition receptors. Furthermore, compromised stomatal and/or apoplastic defenses were observed in mutants of several fucosyltransferases with specific substrates . Collectively, these results uncover a novel and broad role of L-fucose and protein fucosylation in plant immuinity.Discovery of  Pseudomonas syringae pv. tomato (Pst) is indirectly perceived by the RESISTANCE TO P. syringae 2 (RPS2) protein and activates downstream ETI signaling and defense outputs, including the hypersensitive cell death response  and antimicrobial compounds, as well as an inducible innate immune system . Researcresponse .P. syringae often go through an initial epiphytic phase on the surfaces of the above-ground parts  of plants and a subsequent endophytic phase inside the apoplast (intercellular spaces between mesophyll cells), where aggressive multiplication takes place  plays multiple roles in ectively . COR is ectively  and otheectively . COR intectively . COR alsectively . In addi defense . Importa defense .Pst DC3000 infection was also demonstrated in an unbiased genetic screen by Zeng and colleagues for Arabidopsis mutants with increased susceptibility toCOR-deficient Pst DC3000 (scord) , GMD2  . Of the mur1-1 (CS6243) (mur2-1 (CS8565) (fut4 (SAIL_284_B05) (fut6 (SALK_099500) (cgl1-3 (SALK_073650) (fucTa (SALK_087481) (fucTb (SALK_063355) (fut13-2 (SALK_067444) (stt3a-2 (SALK_058814) (stt3b-1 (SALK_033391) (spy-3 (CS6268) (spy-5 (CS8094)  , mur2-1 (CS8565) , fut4 (S284_B05) , fut6 (S_099500) , cgl1-3 _073650) , fucTa (_087481) , fucTb (_063355) , fut13-2_067444) , stt3a-2_058814) , stt3b-1_033391) , spy-3 ((CS6268)  and spy-(CS8094) . Arabidoscord6 mutant and the parental Col-7 plant was extracted using PowerPlant Pro DNA Isolation Kit (Mo-Bio) and sent to the Michigan State University (MSU) Research Technology Support Facility (RTSF) Genomic Core for paired-end sequencing using Illumina’s HiSeq 2500 next-generation sequencer. A total of ~3 Gbp genome sequences for each sample were obtained and the coverage was 21- and 26-fold for the scord6 mutant and Col-7 wild-type, respectively. The read quality control was assessed using FastQC  (scord6 mutant were described in Genomic DNA of the g FastQC . The rea pSKI015  using Bo pSKI015 . Sequenc pSKI015 . Homozyg mapping . Only noer (IGV)  and PCR.MUR1 (AT3G51160) gene was amplified from a total mRNA extract of Col-0 leaf tissue and cloned into the donor vector pDONR207 via BP Clonase II (Invitrogen), generating the entry clone. The entry clone was then recombined with the destination vector pGWB517 (35Spro:MUR1-4xMyc. The confirmed construct was introduced into Agrobacterium tumefaciens (GV3101) by electroporation. 35Spro:MUR1-4xMyc construct was then transformed into the scord6 mutant via A. tumefaciens-mediated Arabidopsis transformation (−1 hygromycin was used to select transgenic seedlings expressing the 35Spro:MUR1-4xMyc transgene.The coding DNA sequence (CDS) of the  pGWB517  using LRormation . Half-stP. syringae infection assays in Arabidopsis were performed as described previously  or syringe-infiltrated with a bacterial suspension (Pst DC3118 [1 × 105 ~ 1 × 106 cfu ml−1] or Pst DC3000 (avrRpt2) [1 × 106 cfu ml−1] in 0.25 mM MgCl2 solution). Bacterial populations were determined by serial dilutions of plant homogenates after two or three days post-inoculation. A total of 12 leaf discs (diameter of 4 mm) from three fully expanded leaves of one plant were collected as one biological replicate. For MAMP-induced protection assays, plant leaves were syringe-infiltrated with a 0.1 μM flg22 (EZBiolab), 0.5 μM elf26 (EZBiolab), or 0.1% DMSO (mock) for 22-24 hours followed by infiltration of bacterial suspension (1 × 106 cfu ml−1Pst DC3000 in 0.25 mM MgCl2 solution).eviously . Briefly5 cfu ml−1Pst DC3118  for two hours. Stomatal apertures were captured using Olympus FluoView 1000 Spectral-based Laser Scanning Confocal Microscope with excitation/emission at 405/460 nm. The length and width of the pore aperture were measured using ImageJ (https://imagej.nih.gov/ij/). Stomata from four different plants per genotype  were imaged. At least 30 stomata per genotype were measured for each treatment and the ratio of width/length was graphed to represent the stomatal aperture status.Leaf discs (~3 mm × 3 mm) were collected one hour after lights turned on in the growth chamber and were submerged in MES buffer  with 100 μM SA (Sigma-Aldrich), 10 μM ABA (Sigma-Aldrich) or 0.1% DMSO (mock) for one hour. For measuring pathogen-induced stomatal closure, leaf discs were submerged in water or bacterial suspension (1x10−1 butylated hydroxytoluene supplemented with 100 nM deuterated-ABA (ABA-2H6) as an internal standard. After overnight incubation at 4 °C with gentle agitation, samples were cleared by centrifugation (12000 × g) and filtered through a 0.2 μm PTFE membrane (Millipore) before being transferred to autosampler vials. ABA (Sigma-Aldrich) was used to prepare calibration standards and generate standard curves (3.9 nM-1000 nM). 10 uL injections of plant extracts were separated using an Acquity UPLC module (Waters) equipped with an Ascentis Express fused-core C18 Column  heated to 50 °C. The aqueous phase A consisted of 0.15% formic acid in water and the organic phase B was methanol. The separation consisted of a linear increase from A:B  to 100% B over 2.5 minutes at a flow rate of 0.4 ml minute−1. Transitions from deprotonated molecules to characteristic product ions were monitored for ABA (m/z 263.1>153.1) and ABA-2H6 (m/z 269.1>159.1) on a Quattro Premier tandem mass spectrometer (Waters) in negative ion mode. The capillary voltage was 3500 V, cone voltage 25 V, and extractor voltage 5 V. The source temperature was maintained at 120 °C and desolvation temperature was 350 °C. Nitrogen was used as desolvation and cone gas at a flow rate of 600 l h−1 and 50 l h−1, respectively. The collision energies and source cone potentials were optimized for each transition using QuanOptimize software. MassLynx 4.1 equipped with application manager QuanLynx was used for data acquisition and processing.Phytohormones were extracted and quantified as described previously  with minSEM samples were prepared and scanned at the Center for Advanced Microscopy, MSU. Leaf samples were fixed at 4 °C for one and a half hours in 4% glutaraldehyde buffered with 0.1 M sodium phosphate at pH 7.4. Following a brief rinse in the buffer, samples were dehydrated in an ethanol series  for 50-60 min each, and then in 100% ethanol for one hour three times. Samples were dried in a Leica Microsystems model EM CPD300 critical point dryer (Leica Microsystems) using liquid carbon dioxide as the transitional fluid. Samples were then mounted on aluminum stubs using high vacuum carbon tabs (SPI Supplies) and coated with osmium (~ 10 nm thickness) in an NEOC-AT osmium coater . Finally, samples were examined with a JEOL JSM-6610LV scanning electron microscope with SEI mode at 12 kV accelerating voltage, 30 spot size and ~15 mm working distance (JEOL Ltd.).TEM samples were prepared and scanned at the Center for Advanced Microscopy, MSU. After primary fixation , samples were washed with 0.1 M cacodylate buffer and postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer, dehydrated in a gradient series of acetone and infiltrated and embedded in Spurr. 70-nm thin sections were obtained using a Power Tome Ultramicrotome  and post-stained with uranyl acetate and lead citrate. Images were taken with JEOL 100CXII Transmission Electron Microscope (JEOL Ltd.) at an accelerating voltage of 100kV.35Spro:MUR1-4xMyc plants, rabbit polyclonal anti-c-Myc primary antibodies  and goat anti-rabbit secondary antibodies  were used. For detecting α1,3-fucosylated protein N-glycans, rabbit anti-fucose primary antibodies  and goat anti-rabbit secondary antibodies  were used. For detecting loading controls, the membranes were stained with staining solution  Naphthol Blue Black (Sigma-Aldrich)) for 20 min, and destained twice with destaining solution  for 10 min each.Proteins were extracted from four- to five-week-old plants using protein extraction buffer , 1% protease inhibitor (Sigma-Aldrich) and 1 mM DTT). Protein concentrations were measured using the RC/DC protein assay kit (BioRad) with bovine serum albumin  as standards. Protein samples were adjusted to the same concentrations of total proteins and immunoblotted with the corresponding antibodies. For evaluating the expression of MUR1-4xMyc from −1 tissue) of protein extraction buffer , 0.5% sodium deoxycholate, and 1 x cOmplete mini protease inhibitor ). Crude extracts were subsequently centrifuged at 10,000 x g to pellet insoluble materials. Solubilized protein was normalized via Bradford assay (BioRad). For relative abundance comparisons, approximately 60 ug of solubilized protein was separated by SDS-PAGE on 4-12% NuPAGE bis-tris gels (ThermoFisher). For deglycosylation experiments, 20 ug of solubilized protein was treated with N-glycosidase F (PNGase F) (New England Biolabs) according to the manufacturer’s instructions and separated by SDS-PAGE on 3-8% NuPAGE tris-acetate gels (ThermoFisher). SDS-PAGE gels were transferred to PVDF membranes using an iBlot 2 dry blotting system (ThermoFisher) and proteins were analyzed by immunoblotting with anti-FLS2  and anti-BAK1  antibodies. Horseradish peroxidase-conjugated anti-rabbit IgG antibody  was used as a secondary antibody and visualized with either SuperSignal West Dura or SuperSignal West Pico PLUS chemiluminescent substrates.Immunoblot of FLS2 and BRI1-ASSOCIATED KINASE1 (BAK1) proteins, was performed as previously described  with minscord6 leaves were collected after elf26, flg22, or 0.1% DMSO (mock) treatment. Total RNA was extracted using RNeasy Plant Mini kit (Qiagen). M-MLV Reverse transcriptase (Life Technologies) and SYBR Green master mix (Life Technologies) were used for reverse transcription and real-time PCR. Primers were described in Col-7 and −1 luminol (Sigma-Aldrich), 20 μg ml−1 horseradish peroxidase (Sigma-Aldrich) in water) with 0.1 μM flg22 or 0.3% DMSO (mock) were added into each well and luminescence was measured immediately at 470 nm using SpectraMax L Luminescence Microplate Reader (Molecular Devices).Leaf discs (diameter = 4 mm) from five-week old plants were harvested and floated  in white 96-well microplate filled with 200 μl water per well. Water was removed 16-18 hours later. 100 μl solution . Leaf discs were collected seven hours post inoculation for both bacterial populations and cAMP quantification. Bacterial populations were determined by serial dilutions of plant homogenates. cAMP was extracted and quantified using the Direct cAMP ELISA kit (ENZO) and normalized by total plant protein, which was quantified using a Quickstart Bradford assay (BioRad). In order to eliminate the influence of different bacterial populations, cAMP amounts (pmol cAMP μg−1 protein) were further divided by the corresponding bacterial populations (cfu cm−2) in each plant.Bacterial effector translocation assays were performed and quantified as described previously . Briefly plasmid . In ordescord6 mutant was previously isolated from the activation-tagging T-DNA (pSKI015) insertion lines  or insertions/deletions (INDELs), in the scord6 genome. Using SAMtools and VCFtools, homozygous variations detected only in the scord6 genome, but not in the parental Col-7 genome, were selected and mapped back to the previously identified candidate regions by physical mapping (MUR1 (GMD2 (MUR1 gene)  . The delR1 gene)  and genoMUR1 encodes an isoform of GDP-D-mannose-4,6-dehydratase and catalyzes the first step of the de novo synthesis of GDP-L-fucose from GDP-D-mannose , and in the sugar-mediated modification of plant proteins   . Arabido mutants  exhibit proteins . We used6 leaves ,c. The l protein  and rest6 mutant ,c.scord6 mutant was previously observed to have an abnormal stomatal morphology  or some other physiological processes, which was a significant uncertainty in our previous study of the scord6 mutant , and FLG22-INDUCED RECEPTOR-LIKE KINASE1 , in the scord6 mutant , which cleaves plant N-glycans without α1,3-fucose in the core N-acetylglucosamine  was infiltrated into the apoplast of the scord6 and mur1-1 mutants and their respective wild-type plants. Bacterial populations of Pst DC3000 (avrRpt2) were significantly higher in the scord6 and mur1-1 mutants, compared to their respective wild-type plants  complex, which catalyzes the transfer of the pre-assembled oligosaccharide to an asparagine residue  initiates the formation of complex and hybrid N-glycans in the Golgi   , also ex(cgl1-3) . These rO-glycan play a role in Arabidopsis immunity. As no pectin-specific fucosyltransferase has been found so far, disease assays and stomatal assays were conducted with Arabidopsis mutants mur2-1  does not affect Arabidopsis defenses, whereas a defect in fucosylation of O-glycan (in the fut4 fut6 mutant) affects apoplastic but not stomatal defense.We next carried out experiments to determine whether defects in fucosylation of cell wall polymers or culation ; S10b. Hculation -d. TheseO-fucosyltransferase, SPINDLY (SPY), was shown to catalyze mono-O-fucosylation (attaching mono fucose to specific Ser/Thr residues) of DELLA transcriptional repressors , which exhibited only moderately enhanced disease susceptibility. This finding suggests that lack of SPY-mediated mono-O-fucosylation of proteins might be a significant contributor to compromised Arabidopsis immunity in the scord6 and mur1-1 mutants.Recently, a protein pressors . This fupressors . Interese plants . Hence, nd spy-5 . Based odefenses , compareSCORD6 gene and provided multiple lines of evidence that SCORD6 is in fact MUR1. Prior to this study, MUR1 has been known as an isoform of GDP-D-mannose-4,6-dehydratase catalyzing the de novo biosynthesis of L-fucose and being involved in fucosylation of xyloglucan, pectin, glycoproteins and DELLA proteins , which was attributed to a defect in the physical barrier of the primary cell wall  showed compromised defense against Pst DC3118 in both dip- and infiltration-inoculations . We observed delayed/reduced expression of PTI early response genes and compromised ROS burst in the ectively . We founectively . An N-glculation . In thisulations  and thate GlcNAc . However protein , but we e mutant ,d.It is worth noting that possible structural changes in cell wall, due to lack of fucose, might have an impact on plant immune signaling. For example, FERONIA (FER), the malectin-like receptor kinase, maintains cell-wall integrity during salt stress and appears to act as a scaffold to modulate FLS2-BAK1 receptor kinase complex assembly and plant immunity . Moreovespy mutants. In particular, the immune defect in the spy mutant against Pst DC3118 infection was severe and comparable to that observed for the mur1-1 mutant (fucTa fucTb and fut4 fut6) tested in our study only exhibited moderately enhanced bacterial growth, it is likely that SPY-mediated mono-O-fucosylation of proteins plays a critical role in mediating plant immunity. Although SPY-mediated mono-O-fucosylation activates DELLAs and in turn suppresses GA signaling (Pst DC3118 of spy mutants probably result from a defect in a pathway other than GA signaling, because a quintuple mutant of DELLA proteins, della (Pst DC3118, similar to wild-type Ler plants (Pst DC3000 was detected in the quadruple DELLA (P. syringae. These findings therefore raise the possibility that the SPY protein mediates plant immunity through pathways other than GA signaling, and besides DELLAs, there are likely other substrates of the SPY protein.Another interesting observation made in this study is the significantly compromised plant immunity in 1 mutant . As otheignaling , the incs, della , showed r plants . Moreovele DELLA , furtherSCORD6 as MUR1 led us to uncover a novel and multifaceted role of L-fucose biosynthesis and protein fucosylation genes in plant immunity against bacterial pathogen P. syringae and connect L-fucose to central plant immunity pathways, including PTI and ETI. These results open new avenues for future studies to identify immune-related targets of fucosylation and has potential to reveal new insights into immune-related polysaccharides or proteins in plants.In summary, identification of Supplementary MaterialPst DC3118.Fig. S1: Bacterial populations 1 hour after infiltration-inoculation with scord6 mutant.Fig. S2: Loss of Basta resistance in the Fig. S3: SEM and TEM images of stomatal apertures.MUR1 gene affect pathogen- and SA-induced stomatal closure in Arabidopsis.Fig. S4: Mutations in the scord6 mutant.Fig. S5: ABA levels in Col-7 and the MUR1 gene affect Arabidopsis apoplastic defense.Fig. S6: Mutations in the scord6 mutant plants.Fig. S7: Bacterial effector translocation in Col-7 and the Fig. S8: FLS2 and BAK1 abundance and sensitivity to PNGase F.N-glycan processing and modification of O-glycan and xyloglucan.Fig. S9: Simplified diagrams of della mutant.Fig. S10: Disease assays of Arabidopsis mutants of fucosyltransferases and quintuple Table S1: List of primers.Table S2: Summary of key assay results of the Arabidopsis mutants analyzed in this study.


K-RAS mutations occur in approximately 25% of human lung cancers and are most frequently found in codon 12 . Mutated K-RAS inhibitors have shown beneficial results in many patients; however, the inhibitors specifically target K-RASG12C and acquired resistance is a common occurrence. Therefore, new treatments targeting all kinds of oncogenic K-RAS mutations with a durable response are needed. RUNX3 acts as a pioneer factor of the restriction (R)-point, which is critical for the life and death of cells. RUNX3 is inactivated in most K-RAS-activated mouse and human lung cancers. Deletion of mouse lung Runx3 induces adenomas (ADs) and facilitates the development of K-Ras-activated adenocarcinomas (ADCs). In this study, conditional restoration of Runx3 in an established K-Ras-activated mouse lung cancer model regressed both ADs and ADCs and suppressed cancer recurrence, markedly increasing mouse survival. Runx3 restoration suppressed K-Ras-activated lung cancer mainly through Arf-p53 pathway-mediated apoptosis and partly through p53-independent inhibition of proliferation. This study provides in vivo evidence supporting RUNX3 as a therapeutic tool for the treatment of K-RAS-activated lung cancers with a durable response.Oncogenic  K-RAS gene [K-RAS mutation (G12CK-RAS) were conditionally approved by the USA Food and Drug Administration [G12CK-RAS-mutated lung cancers. However, the cancers commonly recur within 1 year. The acquired drug resistance is mainly due to secondary oncogenic mutations occurring at oncogenic K-RAS itself or at both upstream  and downstream (MAPK/MEK pathway) sites [K-Ras but recurred after 2 weeks with secondary oncogene activation [Lung adenocarcinoma (ADC) is the most frequent subtype of lung cancer. Most lung ADCs develop through stepwise progression from adenoma (AD) to ADCs ,2. ApproRAS gene . The mosstration . The inhy) sites ,6,7. Thitivation . These rK-Ras involving the Arf-p53 pathway [K-Ras and inactivation of p53 in the mouse lung accelerates malignant progression to ADC [p53 is considered an attractive therapeutic intervention. However, p53 restoration in K-Ras-activated mouse lung cancer suppresses ADCs but does not affect ADs, which are likely to develop into ADCs [K-Ras mutations induce lung AD/ADC in the absence of p53 mutation [p53 does not have a significant impact on early K-Ras-induced lung tumorigenesis [K-Ras mutations remains unclear.To establish new treatments with a durable response, it is important to determine whether cells have evolved effective defense mechanisms against oncogene activation. Normal cells have a defense mechanism against oncogenic  pathway ,10,11. Sn to ADC . Considento ADCs ,14,15. Cmutation , and losigenesis . WhetherRUNX3 is deactivated by epigenetic alterations in most cases of K-RAS-activated mouse and human lung ADCs [Runx3 inactivation not only abrogates R-point-associated Arf-p53 pathway activity but also promotes the formation of lung ADs and accelerates the development of oncogenic K-Ras-dependent ADCs [A tumor is defined as an abnormal mass of tissue that forms when cells divide at a higher rate than normal or do not die when they should. The cellular decision regarding whether to undergo division or death is made at the restriction (R)-point, which is disrupted in nearly all tumors . RUNX3 fung ADCs ,25. RunxRunx3 inactivation are unable to defend against oncogenic K-Ras, resulting in the transition from AD to ADC upon oncogenic K-Ras mutation. However, it remains unclear whether Runx3 can effectively regress already-established K-Ras-activated lung AD and ADC in vivo. To address this question, we developed a mouse model in which Runx3 is conditionally restored by inducible Flippase. This mouse model was used to demonstrate that Runx3 restoration effectively regresses established lung cancers and inhibits recurrence. The results indicate that cells have evolved an effective defense mechanism against heterozygous oncogenic K-Ras mutation. The mechanism is abrogated by Runx3 inactivation and can be re-established with Runx3 restoration. These results support the potential of RUNX3 as a therapeutic tool for the treatment of K-RAS-activated lung cancers with a durable response.Conceivably, lung AD cells that develop as a result of floxRunx3 (Jax 008773), floxp53 (Jax 008462), LSL-G12DK-Ras (Jax 008179), LA1K-Ras , Rosa26R-Tomato (Jax 007914), and ERT2Flp  mice were obtained from Jackson Laboratory . Frt-Stop-Frt/+Runx3 (FSF/+Runx3) mice originated from Macrogen . All mice analyzed had mixed genetic backgrounds and were age matched (6–8 weeks old) unless mentioned specifically. Sample size was determined based on our experience and previous experiments. No data were excluded from analysis. Animal experiments described were repeated with at least three independent replicates with significant results in the same direction as those represented in the figures. All animal studies were randomized in ‘control’ or ‘treated’ groups. However, all animals housed within the same cage were generally placed within the same treatment group. For analysis of tumor samples, identities were blinded from histopathological assessment. All animals were housed in SPF (Specific Pathogen Free) facilities. The animal studies were approved by the Institutional Animal Care Committee of Chungbuk National University.Ad-Cre) was purchased from Vector Biolabs . Each mouse was treated with a titer of 2.5 × 107 Ad-Cre viral genome copies diluted in 50 μL warm sterile MEM. After the treatment, mice were placed on a warm pad until they woke up. Tamoxifen-containing food was administered every day at 400 mg/kg food . Animal studies were approved by the Institutional Animal Care Committee of Chungbuk National University. Animals were maintained under specific pathogen-free conditions and monitored daily.Adenovirus carrying Cre recombinase  and nasally infected them with Ad-Cre to induce oncogenic K-Ras-dependent lung cancer. Four weeks after the virus infection, we sacrificed the mice and extracted their lungs to obtain the KPR primary tumor cell line. The tumor burden from the lung was sliced into tiny pieces and treated with trypsin-EDTA so that the KPR primary tumor cells were isolated. The cells were stabilized in 20% FBS-containing DMEM for days and cultured in 10% FBS-containing DMEM.We prepared 6- to 8-week-old H&E staining experiments were performed following standard protocols. Briefly, slides were rehydrated with ethanol, xylene, and water to remove the paraffin. The nuclei were stained with hematoxylin  for 3 min and the cytoplasm was stained with eosin  for 30 seconds. Slides were mounted with Permount  after the dehydration and clearing steps.https://www.zeiss.com.cn/microscopy/products/microscope-software/zen-lite/zen-lite-download.html accessed on 6 October 2023.For histological analyses, lungs were inflated with 4% paraformaldehyde or formalin  and fixed for 36 h. Fixed paraffin sections were rehydrated, subjected to antigen retrieval, blocked in TBS (0.1% Triton X-100 containing 1% BSA) or DAKO protein-free blocking solution, and sequentially incubated with specific primary antibodies, biotinylated secondary antibodies (DAKO), and the Alexa Fluor system . Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed using the in situ Cell Death Detection kit (Roche). Images were produced using a conventional microscope mounted with a DP71 digital camera (Olympus), an LSM 710 T-PMT confocal microscope (Carl Zeiss), and an AXIO Zoom.V16 and ApoTome.2 (Carl Zeiss). Images were processed with equivalent parameters using the ZEN Light Edition software (Carl Zeiss) For the generation of standard exome capture libraries, the Agilent SureSelect Target Enrichment protocol for the Illumina paired-end sequencing library  was used with 1 µg input gDNA. In all cases, the SureSelect Human All Exon V6 or SureSelect Mouse All Exon probe set was used. DNA quantification and DNA quality were analyzed using PicoGreen and agarose gel electrophoresis. One microgram of genomic DNA from each cell line was diluted in EB buffer and sheared to a target peak size of 150–200 bp using the Covaris LE220 focused ultrasonicator  according to the manufacturer’s recommendations. The 8 microTUBE Strip was loaded into the tube holder of the ultrasonicator, and the DNA was sheared using the following settings: mode, frequency sweeping; duty cycle, 10%; intensity, 5; cycles per burst, 200; duration, 60 sec × 6 cycles; and temperature, 4–7 °C. The fragmented DNA was repaired, an ‘A’ was ligated to the 3′ end, and Agilent adapters were then ligated to the fragments. Once ligation was assessed, the adapter-ligated product was PCR amplified. The final purified product was quantified using the TapeStation DNA screentape D1000 . For exome capture, 250 ng of the DNA library was mixed with hybridization buffers, blocking mixes, RNase block, and 5 µL of SureSelect all-exon capture library, according to the standard Agilent SureSelect Target Enrichment protocol. Hybridization to the capture baits was performed at 65 °C using the heated thermal cycler lid option at 105 °C for 24 h on a PCR machine. The captured DNA was then washed and amplified. The final purified product was quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms), analyzed using the TapeStation DNA screentape D1000 (Agilent), and sequenced using the HiSeq™ 2500 platform .Transient transfections in all cell lines were performed using Lipofectamine Plus reagent and Lipofectamine (Invitrogen). Cell lysates were incubated with the appropriate mono- or polyclonal antibodies (2 μg antibody/500 μg lysate sample) for 3 h at 4 °C, and then with protein G–Sepharose beads  for 1 h at 4 °C. For detection of endogenous proteins, lysates were incubated with the appropriate mono- or polyclonal antibodies (dilution range 1:1000–1:3000) for 6–12 h at 4 °C, and then with protein G–Sepharose beads (Amersham Pharmacia Biotech) for 3 h at 4 °C. Immunoprecipitates were resolved using SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane . The membrane was immunoblotted with the appropriate antibodies after blocking and visualized on an Amersham™ Imager 600  after treatment with ECL solution (Amersham Pharmacia Biotech).Antibodies targeting p300 (Cat# sc-584), p53 (Cat# sc-126), Arf , Tbp (Cat# sc-421), and Brg-1  were obtained from Santa Cruz Biotechnology . Antibodies targeting Brd2 (Cat# H00006046-M01) were obtained from Abnova . All antibodies were diluted to 1:1000. Antibodies targeting Runx3 (Cat# ab40278) were obtained from Abcam  and diluted to 1:3000.Total RNA was isolated using Trizol reagent (Invitrogen). RNA quality was assessed using an Agilent 2100 bioanalyzer  and RNA quantification was performed using an ND-2000 Spectrophotometer . Libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA-Seq Kit . Isolation of mRNA was performed using the Poly(A) RNA Selection Kit . The isolated mRNAs were used for cDNA synthesis and shearing in accordance with the manufacturer’s instructions. Indexing was performed using the Illumina indexes 1–12. The enrichment step was carried out using PCR. Subsequently, libraries were checked using the TapeStation HS D1000 Screen Tape  to evaluate the mean fragment size. Quantification was performed using the library quantification kit and a StepOne Real-Time PCR System . High-throughput sequencing was performed as paired-end 100 sequencing using NovaSeq 6000 .Quality control of raw sequencing data was performed using FastQC . AdapterRunx3 in K-Ras-activated lung ADCs, we developed a mouse model, Frt-Stop-FrtRunx3 (hereafter FSFRunx3), in which Runx3 is deactivated by a Frt-Stop-Frt cassette, but can be conditionally restored via deletion of the cassette through the activation of Flippase recombinase (Flp)  . Runx3FS−/− mice , died wiFSF/+Runx3 mice were crossed with strains harboring Rosa26R-Tomato (Tomato*), LoxP-Stop-LoxP-G12D/+K-Ras, flox/floxRunx3, and ERT2Flp, yielding Tomato*, LoxP-Stop-LoxP-G12D/+K-Ras, flox/FSFRunx3, and ERT2Flp mice (KRL/F mice)  transduced by Ad-Cre (adenovirus carrying CMV promoter-driven Cre recombinase) activates LoxP-Stop-LoxP-G12DK-Ras and deactivates floxRunx3, and treatment with tamoxifen (TAM) restores Runx3 by activating ERT2Flp (/F mice) B. A Toma FlpERT2 B.Runx3 restoration could regress already-established K-Ras-activated lung ADs and/or ADCs, the KRL/F mice were infected with Ad-Cre through nasal inhalation (2.5 × 107 pfu/mouse) [K-Ras activation and Runx3 inactivation. Six weeks after Ad-Cre infection, the lungs of the mice exhibited Tomato fluorescence under UV light, indicating tumor development (L/F-TAM(-), n = 5) or tamoxifen-containing food (KRL/F-TAM(+), n = 5) to promote Runx3 restoration. KRL/F-TAM(-) mice survived for an average of 13.2 weeks after infection, and all died by 14 weeks after infection (L/F-TAM(+) mice survived for an average of 28.2 weeks after infection, and all died by 30 weeks after infection  ,25 for Kelopment C. Microselopment C. The renfection D. In connfection D. These L/F-TAM(-) and KRL/F-TAM(+) mice were sacrificed at 4 or 10 weeks after tamoxifen treatment (L/F-TAM(-)-4w mouse lungs exhibited high levels of Tomato fluorescence under UV light and developed large lung ADs/ADCs (L/F-TAM(+)-4w mice exhibited very low levels of Tomato fluorescence under UV light -4w and KRL/F-TAM(+)-10w mice suggest that nearly all the K-Ras-activated lung AD cells and ADC cells were eliminated through Runx3 restoration. Genotyping of the lung tumors confirmed K-Ras activation, Runx3 inactivation by Ad-Cre infection, and Runx3 restoration via tamoxifen treatment  staining of the mouse lungs showed that the Tomato-positive lung cancer cells of KRL/F-TAM(-)-1w mice were TUNEL-negative. However, most of the Tomato-positive lung cancer cells of KRL/F-TAM(+)-1w mice were TUNEL-positive (Runx3-restored cells underwent apoptosis. Enlarged microscopic images of the figure are shown in Arf is a major target of Runx3 [Runx3 restoration induced Arf and p53 expression in the lung cancers (L/F-TAM(+)-4w mice contained a few lesions that were undergoing regression, and the cells in these lesions were TUNEL-positive  (L/F-TAM(+) mice were sacrificed at 14 weeks after tamoxifen treatment (L/F-TAM(+)-14w mouse lungs showed that small ADCs had developed in all four mice (K-Ras-activated cells (K-Ras was mutated (G12DK-Ras) in KRL/F-TAM(-)-0w mouse lung ADCs and recurrent lung ADCs, as targeted in the LSL-G12DK-Ras allele. There were no additional mutations in K-Ras, and none of the other known major oncogenes  or tumor suppressors  involved in lung cancer were mutated in any of the ADCs (All of the fection) D. In a preatment D. Analysour mice A. The ADed cells A. Whole-the ADCs . This suFSFRunx3 allele by tamoxifen-activated ERT2Flp  and LA1/+; Runx3FSF/+K-Ras; and ERT2Flp (KLA1RFSF/+) mice  is deactivated by the Frt-Stop-Frt cassette. The FSFRunx3 allele can be restored by tamoxifen, which activates ERT2Flp.g cancer . To unde/+) mice A. In theLA1R+/+ mice and KLA1RFSF/+ mice in the absence of tamoxifen. The KLA1R+/+ mice (n = 15) developed lung cancer and began to die at 26 weeks after birth, and all the mice died within 72 weeks after birth  (LA1RFSF/+ mice (n = 18) began to die at 10 weeks after birth, and all the mice died within 70 weeks after birth  .We measured the lifespan of K7 weeks) B,C. The /+ mice) B,C. ThesLA1RFSF/+ mice were fed tamoxifen-containing food for Runx3 restoration for 2 weeks starting at 10 weeks after birth (KLA1RFSF/+-TAM(+), n = 15). Runx3 restoration significantly extended the survival of the KLA1RFSF/+ mice: KLA1RFSF/+-TAM(+) mice survived for an average of 52.9 weeks after birth, which is 13.4 weeks longer than the KLA1RFSF/+-TAM(-) mice (p = 0.02) (LA1RFSF/+-TAM(+) mice were similar to that of KLA1R+/+ mice (LA1RFSF/+-TAM(-) mice and KLA1RFSF/+-TAM(+) mice revealed that Runx3 was silenced by CpG island hyper-methylation in all the analyzed lung ADCs  B,C. The +/+ mice B,C. MS-Pung ADCs D,E. Immuung ADCs F. These as alone .Runx3 inactivation is essential for the maintenance of K-Ras-activated lung cancer, the survival of the model mice for activation via K-Ras alone should be directly related to the number of functional Runx3 alleles. KLA1R+/+ mice have two functional Runx3 alleles. KLA1RFSF/+-TAM(-) mice have only one functional Runx3 allele because the other allele is inactivated. KLA1RFSF/+-TAM(+) mice have two functional Runx3 alleles because the inactivated Runx3 allele is restored. The survival of the mouse models was indeed directly related to the number of functional Runx3 alleles , and KLA1RFSF/+-TAM(+) mice was confirmed through immunoblotting (IB) using a K-RasG12D-specific antibody (Runx3 in the lung ADCs developed in KLA1RFSF/+-TAM(+) mice was confirmed with genomic DNA PCR . In KPRL/F mice, Ad-Cre infection activated LSL-G12DK-Ras, deactivated floxp53 and floxRunx3, and labeled the targeted cells with Tomato fluorescence  (L/F mice was 13.2 weeks (L/F mice was approximately 3.4 weeks shorter than that of KRL/F mice (p = 0.0018).Two weeks after g cancer B. MicrosL/F mice C. The Ad8 weeks) D. The me.2 weeks D. These L/F mice ) or tamoxifen-containing food (KPRL/F-TAM(+)) for 2 weeks (L/F-TAM(-) mice and 14.2 weeks in KPRL/F-TAM(+) mice . Although Runx3 restoration significantly extended the survival of KPRL/F mice (≈4.4 weeks), a comparison of the effect of Runx3 restoration with that on KRL/F mice (>18 weeks) (After confirming tumor development in KPR 2 weeks E. The me(+) mice E. These 8 weeks) D indicatL/F-TAM(-) and KPRL/F-TAM(+) lung cancers confirmed targeting of LSL-G12DK-Ras, floxp53, and floxRunx3 alleles by Ad-Cre infection  cancers, indicating that Runx3 was restored in these cancers  and KPRL/F-TAM(+) lung cancers, which bear a heterozygous oncogenic K-Ras mutation. The results of immunostaining showed that Arf was not expressed in KPRL/F-TAM(-) lung cancers, whereas it was expressed in KPRL/F-TAM(+) lung cancers in which Runx3 was restored  complex, which induces ARF expression in response to oncogenic K-RAS activity . The Rpa unclear ,14,15. Trestored . These rK-Ras mutation, we obtained immortalized cell lines from KPRL/F-TAM(-) lung cancers (KPR-) (- cell lines with 4-hydroxytamoxifen (4-OHT) restored Runx3, generating KPRrestored cell lines   F. Treatmll lines . We confreatment G. Immunoreatment G. The Rpestored) G. These - and KPRrestored cell lines were transfected with empty vector or p53-expressing plasmid. Ectopic expression of p53 in the KPR- cells, in which Arf was not expressed, activated Caspase-3 weakly  mice and KPRL/F-TAM(+) mice were pathologically indistinguishable (restored cells was lower than that of KPR- cells (p = 0.003) (L/F lung tumors was significantly reduced with Runx3 restoration (KPRL/F-TAM(-) = 797.2/mm2; KPRL/F-TAM(+) = 423.5/mm2, p = 0.023)  A. Consis= 0.023) B,C. EnlaRunx3 in the K-Ras-activated lung cancer cells, we performed mRNA sequencing (RNA-seq) in KPR- cells and KPRrestored cells. Analysis of the Z-scores revealed that 3,194 and 3,002 genes were induced and suppressed, respectively, in response to Runx3 restoration . We previously reported that inactivation of Runx3 in the mouse lung induces the development of ADs [Runx3 inactivation is an earlier event than K-Ras activation in a carcinogen-induced mouse lung cancer model that recapitulates the features of K-RAS-driven human lung cancers [Runx3, results in both AD development and the disruption of the Arf-p53 pathway. Indeed, K-Ras activation, with or without p53 inactivation, in an extremely small number of cells failed to induce pathologic lesions for up to 1 year [Runx3 inactivation and K-Ras activation with the same targeting method led to the rapid induction of lung ADs and ADCs, and it caused lethality in all the targeted mice within 3 months [Runx3 restoration may regress K-Ras-mutated lung tumors and result in sustained regression without recurrence, similar to the effect of Apc restoration. In this study, Runx3 restoration in a mouse lung cancer model regressed K-Ras-activated ADs as well as ADCs, suppressed secondary oncogene activation, and markedly extended the survival of mice (by approximately 15 weeks). Knockdown of oncogenic K-Ras regresses mouse lung cancer; however, the lung cancer recurs after 2 weeks with secondary oncogene activation [Runx3-restored mouse lung cancer did not recur until 10 weeks after regression (14 weeks after Runx3 restoration). The recurred tumor demonstrated spontaneous inactivation of the restored Runx3 allele without secondary oncogene activation. These results show that expression of Runx3 could be helpful for the treatment of lung cancer and for achieving sustained regression.However, another possibility was suggested. The initial step of colorectal AD development is the inactivation of adenomatous polyposis coli (APC), and activation of elopment ,38. In acurrence . These rt of ADs ,25. In a cancers . Runx3 p cancers . Therefoo 1 year . In cont3 months . TherefoK-Ras-activated lung cancer was stopped upon Runx3 inactivation and recovered with Runx3 restoration  is suppressed through Runx3 inactivation. In contrast, Runx3 restoration recovers the oncogene surveillance mechanism and could, therefore, lead to inhibition of secondary oncogene activation as well as cancer regression. It has long remained unclear why the Arf-p53 pathway fails to eliminate K-Ras-activated lung ADs [Runx3 silencing in lung ADs. This does not explain why the p53 mutation still occurs after K-RAS activation in lung tumorigenesis. The ARF-p53 pathway protects cells from oncogene activation. In contrast, the ATM/ATR-p53 pathway protects cells from genome instability [Arf expression in toration . The tumtoration D. Taken lung ADs ,14. The tability . TherefoK-RAS-activated lung cancers, but in almost all other malignancies, clinical responses have been yielded through the application of targeted therapies that inhibit activated oncogenes, but despite the application of these therapies, tumor recurrence has eventually resulted [Runx3 as such a tumor suppressor that could effectively regress established K-Ras-activated mouse lung cancer and inhibit cancer recurrence.Not only in resulted ,43. Ther


Skin employs interdependent cellular networks to facilitate barrier integrity and host immunity through ill-defined mechanisms. This study demonstrates that manipulation of itch-sensing neurons bearing the Mas-related G protein-coupled receptor A3 (MrgprA3) drives IL-17+ γδ T cell expansion, epidermal thickening, and resistance to the human pathogen Schistosoma mansoni through mechanisms that require myeloid antigen presenting cells (APC). Activated MrgprA3 neurons instruct myeloid APCs to downregulate interleukin 33 (IL-33) and up-regulate TNFα partially through the neuropeptide calcitonin gene related peptide (CGRP). Strikingly, cell-intrinsic deletion of IL-33 in myeloid APC basally alters chromatin accessibility at inflammatory cytokine loci and promotes IL-17/23-dependent epidermal thickening, keratinocyte hyperplasia, and resistance to helminth infection. Our findings reveal a previously undescribed mechanism of intercellular cross-talk wherein “itch” neuron activation reshapes myeloid cytokine expression patterns to alter skin composition for cutaneous immunity against invasive pathogens.


This review provides recent developments in the current status and latest synthetic methodologies of biphenyl derivatives. Furthermore, this review investigates detailed discussions of several metalated chemical reactions related to biphenyl scaffolds such as Wurtz–Fittig, Ullmann, Bennett–Turner, Negishi, Kumada, Stille, Suzuki–Miyaura, Friedel–Crafts, cyanation, amination, and various electrophilic substitution reactions supported by their mechanistic pathways. Furthermore, the preconditions required for the existence of axial chirality in biaryl compounds are discussed. Furthermore, atropisomerism as a type of axial chirality in biphenyl molecules is discussed. Additionally, this review covers a wide range of biological and medicinal applications of the synthesized compounds involving patented approaches in the last decade corresponding to investigating the crucial role of the biphenyl structures in APIs. This review provides recent developments in the current status and latest synthetic methodologies of biphenyl derivatives. Whereby, Ullmann described the Cu-catalyzed homocoupling reaction involving halo-arenes in 1901. In 1914, Bennett and Turner demonstrated the homodimerization of Grignard reagent and phenyl magnesium bromide which promoted by chromium(iii) chloride (CrCl3) or anhydrous cupric chloride (CuCl2).17 Kumada depicted the reaction of aryl halides with arylmagnesium halide or organozinc in the presence of transition metal catalysts.18–20 Whereas, Hiyama produced biaryls via coupling the readily available organosilanes with organohalides.21,22 In the same context, Negishi et al.23 reported a regio- and chemoselective method for biaryl synthesis.24,25 In 1986, a new methodology for cross coupling reaction of organic electrophiles with organostannanes was discovered by Stille.26,27 After that, Suzuki–Miyaura cross coupling became one of the most effective and widely used ways for forming carbon–carbon bonds.15,28,29 As biphenyl scaffolds are a key step to produce bioactive molecules used for medicine. In addition, biphenyl compounds were isolated by distillation from natural sources such as crude oil, natural gas, and coal tar.6 Several methods are available in the literature for the synthesis of substituted biphenyl scaffolds through using various chemical building blocks according to the following reaction routes:Wurtz reported the first trial for carbon–carbon bond formation reactions between two alkyl halides in the presence of sodium metal. Then, Fitting expanded this work to include the C(sp2.13)–C(sp3) bond formation through using the sodium-mediate coupling reaction of two alkyl halides.17 In 1862, the reaction was extended by Fittig to include coupling between aryl anion and an electrophile such as aryl halide in the presence of various metal surface such as Na, Li, Au, Cu and Ag.30 Wurtz–Fittig reaction could be achieved via the free radical mechanism31 in three steps; dehalogenation, followed by diffusion of the dehalogenated molecules, and finally, coupling process between two aryl halides 1 to afford biphenyl derivative 2.32 It is worthy to mention that, bulky substitution group leads to kinetically prevent Wurtz–Fittig coupling as one of its drawbacks.33 Also, Wurtz–Fittig reaction plays an important role in the synthesis of thermoplastics polymer synthesis (polyphenylenes and oligophenylenes)  .342.235 as it has attracted the attention of many chemists trying to synthesize chiral substituted biphenyl. Moreover, the Ullmann reaction usually is achieved at lower temperatures and shorter time frame through coupling the aryl halide 1 in the presence of powdered copper or nickel catalysts, but it has changed in recent years.36,37 It is worth mentioning that, the degree of stability of substituted chiral biphenyl depends on the nature of the ortho substituents when this asymmetric Ullman coupling.38 Whereas, enantioselective biphenyls 2 were obtained via the reaction of ortho-chlorinated benzaldehyde derivatives 1 in the presence of Ni catalyst  and CrCl3 in diethyl ether (Et2O), incidental to an attempt to prepare organochromium compound.41 Bennett–Turner reaction can be achieved by using CuCl2.42 In this context, noteworthy is the contribution of Krizewsky with Turner using a CuCl2 promoted homocoupling reaction instead of CrCl3  in the presence of O2 gas produced a mixture of biphenyl coupling product 5 (56%) and the corresponding phenol 6 (38%). The proposed mechanism involves in situ deprotonation with oxidative dimerization has been studied by Do et al.44 The production of corresponding phenol was reasonable through trapping of the formed aryl lithium with oxygen gave rise to an aryl anion species which was less prone to oxygenation and more likely to form a copper adduct that would undergo oxidative coupling reaction  with CuClreaction .i) and Cu(ii)) as they are able to mediate the dimerization of arylboronic acids 7 to afford the corresponding symmetrical substituted biaryl 2 in good yields. The reaction can be possibly catalyzed under an oxygen atmosphere without a significant loss of yields.45 In 2009, Kirai et al.46 improved the previous protocol for oxidative dimerization which relying on catalysts that would facilitate transmetalation. As they reported a new effective methodology for homocoupling of aryl boronic acid derivatives 7 under stirring conditions through using the catalytic amount of [{(phen)Cu(μ-OH)}2Cl2]·3H2O. It is worthy to mention that the homocoupling reaction proceeds without any additives such as oxidant or base. Besides, this method tolerates different substituents on the arylboronic acids such as nitro group, halogens and carbonyls  complex 2A to yield bimetallic arylcopper(ii) intermediate 2B without using any base. Biaryl products 2 afford through the concomitant one-electron reduction of each copper center [2LCuII–Ar → 2LCuI + Ar–Ar]. As the binuclear copper models efficiently activate molecular oxygen, in the same time the molecular oxygen smoothly bind to the resulting bimetallic intermediate [{(phen)CuI(μ-Cl)}2] 2C, which is reoxidized to afford (μ-hydroxido)copper(ii) complex 2A coppermagnesium bromide 9 in the presence of Ni complex and THF afforded 4′-methoxy-2-methyl-biphenyl 10 in (87%) yield.49 Whereas, in the presence of Pd catalyst, product 10 produced in (94%) yield  yield .50ii) into a close C–H bond. Primary imine derivatives 12 underwent a closure under oxygen-mediated elimination affording the respective phenanthridine derivatives 13  (AgSbF6), silver oxide (Ag2O) and TBAF produced biphenyl-2-carbonitrile derivative 16.56 Whereby, refluxing a mixture of arylsilane 14 with arylhalide 1 in a mixture of AcOH and toluene, Pd catalyst, and tetrabutylammonium fluoride (TBAF) as a base afforded biphenyl 2. Furthermore, this method was applied under mild condition and tolerated high functional groups  to Pd(ii). Then, transmetallation step occurred in the presence of base such as  which encouraged the C–Si bond to split and establish a new C–Pd bond.53,60 Finally, in the reductive elimination step a new C–C bond was formed and Pd returned back to the oxidation state (0) (24Noor and coworkerstate (0) .242.661 SMC reaction is also used to tag DNA with luminous intercalator substituents for DNA sequencing and diagnostics.62 In 1979, Akira Suzuki, Norio Miyaura and Kinji Yamada reported SMC which is the most common method in the formation of carbon–carbon bond in drug discovery.28,29 In the context, the reaction of various aryl bromide derivatives 1 with commercially available aryl boronic acids 7 under action of palladium-catalyzed Suzuki coupling reaction on porous carbon nanospheres (Pd/CNS) afforded biphenyl derivatives 2. It is worthy to mention that, using (Pd/CNS) as catalyst is preferable than other Pd salts due to its highly effective, and lower cost  has witnessed an increase interest due to its widespread academic and industrial application in the production of fine chemicals, polymers, materials, and pharmaceuticals.wer cost .63through three steps.23,64 The first step is the oxidative addition in which organo palladium(ii) complex 2A is formed via coupling of aryl halide 1 with palladium catalyst and this is happening via breaking carbon–halogen bond and by squeezing palladium between the aryl and halogen group. The second step is the transmetalation whereas organoboron compound is converted to nucleophilic borate in the presence of base such as sodium tert-butoxide and carbonate salts then attacking on a Pd(ii) complex 2B to form 2C complex. Finally, reductive elimination step in which the two aryl groups are reductively eliminated from Pd(ii) complex 2C and combine together to form C–C bond and the palladium is reformed  reaction passed reformed .65,666H4)OPPh2) 17 with aryl boronic acids 7 in the presence of bis(dibenzylideneacetone)palladium catalyst (Pd(dba)2), triphenylphosphine (PPh3) (4 equiv.) and potassium phosphate (K3PO4) (2 equiv.) as a base in refluxing dioxane at 105 °C yielded biphenyl phosphine oxide derivatives 18 followed by reduction with a mixture of trichlorosilane and catalytic drops of triethylamine (TEA) produced biphenyl phosphine derivatives 19 diphenylphosphine oxide  with fluorinated phenyl boronic acid 7 in refluxing a mixture of toluene and water with a catalytic amount of palladium acetate (Pd(OAc)2) and bulky phosphine ligand (SPhos) yielded fluorinated aminobiphenyl derivatives 21.69 Analogously, refluxing of 4-chloroaniline (20) with fluorinated phenyl boronic acid 7 with DMF in the presence of catalytic Pd(OAc)2 and K3PO4 as a base at 80 °C afforded fluorinated aminobiphenyl derivatives 21  as a base and tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3) as a catalyst with SPhos as a ligand in refluxing with a mixture of THF, toluene, and H2O. Whereas, using XPhos as a ligand and potassium carbonate (K2CO3) as base afforded fluorinated biphenyl 22 only with an excellent yield (99%) , organic semiconductors, metal–organic frameworks (MOF), and organic polymer of intrinsic microporosity (OMIMs). In addition, organoflourine as substituents of biphenyl have become widespread drug motifs, as they affect nearly adsorption, metabolism, distribution and excretion properties of lead compounds.ld (99%) . The reald (99%) .723PO4 as a base in a refluxing solvent mixture of tetrahydrofuran (THF) and water furnished fluorinated biphenyl 24.6 Additionally, fluorinated biphenyl 26 was synthesized by refluxing iodobenzene 1 and arylboronate 25 in Cu catalyst, phenanthroline ligand (phen) and DMF -1,3-dihydro-2H-imidazol-2-ylidene (IMes) as a ligand.75 Whereas, the reaction of chloromethylbenzene 30 with 27 in the presence of cobalt(ii) chloride (CoCl2) and a ligand/precursor named bis-1H-imidazol-3-ium afforded methylbiphenyl 31  was esterified using methanol (MeOH) with thionyl chloride (SOCl2) to afford 2-amino-3-iodophenyl-propanoate 33. Then, the amino group was protected using tert-butyloxycarbonyl (t-Boc) and sodium bicarbonate (NaHCO3) in MeOH to furnish N-tert-butyloxycarbonyl-3-iodotyrosine methyl ester (34). Refluxing N-tert-butyloxycarbonyl-3-iodotyrosine ester 34 in the presence of Pd(OAc)2, K2CO3 and hetero aryl trifluoroborate salt 35 in MeOH to afford t-butoxycarbonyl-(6-hydroxy-[biphenyl]-yl)propanoate derivatives 36. Finally, deprotection of compounds 36 was achieved via heating them with dil. HCl at 70 °C for 3 h to give the free amino acid 37  through bromination reaction of 2,4,6-trimethoxy acetophenone (38) with ceric ammonium nitrate (CAN) and lithium bromide (LiBr) in acetonitrile (CH3CN), followed by applying SMC of compound 39 with phenylboronic acid (7) in the presence of Pd(PPh3)4 and K2CO3 in a mixture of ethanol (EtOH) and toluene afforded 2,4,6-trimethoxy-ethan-1-one 40 2), and a mixture of H2O and THF afforded methyl-3-bromo-4′-methoxy--2-carboxylate (44). In order to produce biphenyl acetylene derivatives 46, compound 44 was reacted with phenylacetylene derivatives 45 79 (trans retinoic acid (ATRA) analogues with enhanced stability may hold tremendous promise as therapies for large number of cancers and neurological conditions.80 In addition, diphenylacetylene derivatives acted as antimicrobial peptide mimetics.81Treatment of methyl 2-bromo-6-iodobenzoate (43) with (4-methoxyphenyl)boronic acid (7) in the presence of Naes 45 79 . Dipheny82,83 Due to the recent adoption of high-throughput screening (HTS) methods, the field of olefin polymerization facilitated by Ziegler–Natta catalysts is particularly active.84 A significant number of novel nonmetallocene ligands have been discovered using these techniques for polymerization catalysis. Application of SMC of 2,2′-biphenyldiboronic acid 47 with 2-bromoindene 48 in the presence of tetrakis(triphenylphosphine)palladium [Pd(PPh3)4] furnished polycyclic 2,2′-di(1H-inden-2-yl)-1,1′-biphenyl (49) which showed promising activities in olefin metathesis polymerization boronic acid (51) in the presence of palladium catalyst [Pd(PPh3)4] and K3PO4 in refluxing DME/H2O  boronic acid (51) is coupled with  palladium(ii)iodide (52A), followed by intramolecular Heck reaction from 52B to 52C. Finally, Suzuki coupling reaction of  palladium(ii) iodide (52C) with (1H-inden-2-yl) boronic acid (51) to afford 1H-inden-2-yl-dihydrospiro[indene] 52 with 58% yield  was alkylated with 3-bromobenzyl bromide (57) in the presence of sodium iodide (KI) and K2CO3 to furnish fluorinated acid derivative 58. Then, fluorinated acid derivative 58 was reacted with oxalyl chloride (COCl)2 in the presence of a mixture of dimethylformamide and dichloromethane (DMF/DCM) to afford difluorobenzoyl chloride derivative 59. The acid was transformed to benzamide 60 by using oxalyl chloride, then treatment with ammonium carbonate ((NH4)2CO3). Finally, biphenyl benzamide derivatives 61 were produced in a wide range of yields (73–89%) via coupling commercially available boronic acids 7 with compound 60 (87Biphenyl benzamide derivatives inhibit the filamenting temperature-sensitive mutant Z (FtsZ) protein, in addition to having antibacterial activities.pound 60 .87N-Biphenyl pyrrolidine derivatives are employed as ligand in arylaminations that are catalyzed by palladium. Furthermore, 2′-bromo-2-fluoro-5-nitro-1,1′-biphenyl (63) were produced in (81% yield) via Suzuki–Miyaura cross coupling between 2-iodo 4-nitro fluorobenzene (62) and boronic acid 7 in the presence of palladium catalyst and PPh3 in refluxing dioxane. Then, the fluorine atom was nucleophilically substituted by (R)-pyrrolidine (64) in the presence of K2CO3 in DMF to yield 1--N,N-dimethylpyrrolidin-3-amine (65). (R)-[1-(2′-Bromo-5-amino-biphenyl]-2-yl)-pyrrolidin-3-yldimethylamine (66) was produced by refluxing zinc powder and calcium chloride (CaCl2) with compound (R)-65 in EtOH. After that, hypophosphorous acid (H3PO2) was added to compound (R)-66 in a mixture of THF and H2O, followed by treatment with cupper(i)oxide, and sodium nitrite (NaNO2) furnished bromo(biphenyl-2-yl)-dimethylpyrrolidin-3-amine 67. Finally, 2′-(dicyclohexylphosphaneyl)--2-yl-N,N-dimethylpyrrolidin-3-amine 68 was obtained in two steps involving adding n-butyl lithium dropwise to compound (R)-67 in a mixture of THF and hexane as first step. In the second step, chlorodicyclohexylphosphane (Cy2PCl) was added with stirring to replace bromide by dicyclohexylphosphane group  (The synthesis of biphenyl oxazole derivatives 70 was reported by Ahmad e/water) .through alkylation reaction of 1-bromo-3-(chloromethyl)-2-methylbenzene (71) with 3-chloro-4-hydroxybenzaldehyde (72) produced 4-((3-bromo-2-methylbenzyl)oxy)-3-chlorobenzaldehyde (73). Next, refluxing 4-((3-bromo-2-methylbenzyl)oxy)-3-chlorobenzaldehyde (73) with bis(pinacolato)diboron (B2pin2) (74) in 1,4-dioxane and potassium acetate (CH3COOK) in the presence of 1,1′-bis(diphenylphosphino)ferrocene-palladium(ii)dichloride diCrClchloromethane complex [pd(dppf)Cl2CH2Cl2] afforded 3-chloro-4-benzyl)oxy)benzaldehyde (75). Finally, compound 75 was reacted with (3-bromophenyl)isoxazole derivatives 76via Suzuki coupling reaction yielded biphenyl oxazole derivatives 77  (78) in the presence of catalytic amount of palladium grafted on natural asphalt sulfonate Na[Pd-NAS], cesium carbonate (Cs2CO3) as a base in refluxing ethanol furnished biphenyl derivatives 2 Ln is reacted with aryl halide 1 to form Pd(ii) complex 2A. In transmetalation step, Pd(ii) complex 2A cleavage the C–Sn bond of organotin reagent 78 to produce complex 2B. Finally, reductive elimination step for forming C–C bond and the catalyst is regenerated at the same time  (Pd(dba)2) and tris-o-furylphosphine (tfp) furnished biphenyl derivatives 2.98 In 2009, Phapale and Cárdenas99 discovered that Ni-based catalysts (Ni(acac)2/PPh3) facilitate coupling between aryl zinc chlorides 79 and haloarenes 1 affording biphenyl derivatives 2. The reaction of aryl zinc halide 79 and aryl halide 1 in the presence heterogeneous Ni catalyst on charcoal and microwave irradiation afforded biphenyls 2 in acceptable yields (75–95%)  .through three steps. Initially, an organohalide or pseudohalide (such as triflate) is oxidatively added to a low valent metal complex to produce an organometallic derivative with a higher formal oxidation state on the metal center. Then, producing diorganometal species is processed, whereby, the nucleophilic carbon is transmetalate from the nucleophile to the transition metal complex. Finally, a new C–C bond is formed by further reductive elimination, which reproduces the catalytically active species 2− companied with emission of H2 gas. Similarly, the direct dehydrogenative coupling of benzene through low-valent alkaline-earth metals (Ae) intermediates was achieved via formation of metal (Ae) complex with a C6H52− dianion which acts as reducing agent to bulky β-diketiminate ligand BDI 80A to yield [(DIPePBDI)Ca]2(biphenyl) 81 as dianionic N,C-chelating ligand, which rapidly decomposed to afford biphenyl scaffold 2  such as K, Cs or Rb with benzene afforded Caffold 2 .1003.3.1101 Whereby, ionic liquids have already been used in a number of Friedel–Crafts processes, which could significantly reduce the associated environmental concerns.102Friedel–Crafts reactions are subdivided into two fundamental types: alkylation and acylation reactions.3.1.1103 4,4′-Di-tert-butylbiphenyl (82) was synthesized from biphenyl (2), which then, in the presence of catalytic amounts of anhydrous ferric chloride (FeCl3), interacted with tert-butyl chloride in halogenated solvents.104 In the same manner, Friedel–Crafts cycloannulation process produced octamethyl-octahydro-2,2′-binaphthalene 83through adding catalytic amounts of anhydrous AlCl3 to a solution of biphenyl (2) in 1,2-dichloroethane (DCE) followed by dropwise addition of readily available 2,5-dichloro-2,5-dimethylhexane.105 In the same context, a mixture of ammonia and lithium was added dropwisely to a solution of biphenyl (2) in THF till brick red suspension was formed. Then, a quick addition of 1,2-dibromoethane (EDB) was added to afford 2-bromoethyl-1,1′-biphenyl 84.106 A mixture of biphenyl (2), 2-chlorohexane and tributyl phosphine (PBu3) was heated for 6 h to afford 4-(hexan-2-yl)-1,1′-biphenyl (85)  .1033 and DCE at 0 °C for 24 h afforded 4-cyclopentyl-1,1′-biphenyl (87).107 Additionally, using trichloromethane (CHCl3) instead of DCE at 20 °C for 10 h led to formation of 4,4′-dicyclopentyl-1,1′-biphenyl (88)  with cyclopentene (86) in the presence of AlClnyl (88) .1083.1.2109,110The Friedel–Crafts acylation is the reaction of acid anhydrides or acyl chlorides with aromatic compounds in the presence of Lewis acid catalyst for the synthesis of methyl aryl ketone, which are used in pharmaceuticals and fine chemical such as ibuprofen, naproxen, dyes, agrochemicals, and fragrances.3.1.2.1via Friedel–Crafts acylation of biphenyl (2) and succinic anhydride in the presence of AlCl3 and dichloromethane (DCM).111 While, treatment of biphenyl (2) with dihydro-2H-pyran-2,6(3H)-dione in the presence of AlCl3 and nitrobenzene (C6H5NO2) afforded biphenyl-4-yl-5-oxopentanoic acid (90).112 Further, biphenyl-4-carbonyl-benzoic acid (91) was synthesized through free-solvent mechanochemical ball milling method via reaction of biphenyl (2) with phthalic anhydride in the presence of AlCl3.113 Whereas, gaseous boron trifluoride (BF3) was passed through a homogenous mixture of biphenyl (2) and acetic anhydride to synthesize biphenyl-4-yl-2,2-difluoro-6-methyl-dioxaborinine (92) (1144-Phenylbenzoyl-propionic acid (89) can be easily prepared ine (92) .1143.1.2.2iii)oxide (Fe2O3) and acetyl chloride, 1-ethan-1-one (93) was produced.110 In contrast, acetyl chloride in dried carbon disulfide (CS2) was added drop by drop with stirring into a suspension solution containing biphenyl (2), anhydrous AlCl3, and dried (CS2) to obtain 1,1′-bis(ethan-1-one) (94).115 Additionally, biphenyl (2) was refluxed with propionyl chloride for 4 h in DCM containing a catalytic amount of AlCl3 to furnish 1- propan-1-one (95),116 whereas 1,1′-bis(propan-1-one) (96) was produced by refluxing biphenyl (2) with propionyl chloride for 2 h at −5 °C.117 Under anhydrous conditions, a stirred mixture of CS2, biphenyl (2), and anhydrous AlCl3 was treated with pentanoyl chloride to afford 1,1′-biphenyl-4-pentan-1-one (97) (118Reaction of biphenyl (2) with a mixture of activated iron(one (97) .1183 (M = Fe or Al) activating the acetyl chloride to produce an adduct of acyl chloride with MCl3 (93A). This complex is quickly equilibrated with the acylium ion [CH3CO]+[MCl4] (93B), which then attacks biphenyl (2) to produce biphenyl alkyl ketone 93 (102The proposed mechanism for Friedel–Crafts acylation shows that the reaction began with the Lewis acids MCletone 93 .1023.1.2.3via Friedel–Crafts reaction by stirring ethenone (98) with a solution of biphenyl (2) in CS2, then adding AlCl3 ethan-1-one (93) was synthesized ng AlCl3 .1193.1.2.4via the reaction of biphenyl (2) with oxalyl chloride (ClCO)2 in the presence of AlCl3 and DCM.120 While, the reaction of biphenyl (2) with oxalyl chloride under the influence of AlCl3 in DCE produced 1-ethan-1-one (93) and 1,2-diethane-1,2-dione (100)  .1213.2122 Furthermore, a mixture of 4-isopropyl-1,1′-biphenyl (104), 4,4′-diisopropyl-biphenyl (105), and 3,4′-diisopropyl-1,1′-biphenyl (106) were produced through the reaction of biphenyl (2) with propene (101) in the presence of [Fe]-SSZ-24 catalyst (123Reaction of biphenyl with propene (101) in the presence of lithium metal (Li), and THF, followed by hydrolysis afforded a mixture of 4-isopropyl-1,4-dihydro-1,1′-biphenyl (102) and 4-isopropyl-3,4-dihydro-1,1′-biphenyl (103).catalyst .123in situ formation of carbolithiation step in which the biphenyl (2) is doubly reduced in a mixture of lithium metal (Li) and THF affording biphenyl dianion intermediate 102A. Then, biphenyl dianion 102A reacted with propene (101) in THF, followed by addition step to yield intermediate 102B, finally the hydrolysis step is achieved to furnish 4-isopropyl-1,4-dihydro-1,1′-biphenyl (102) and 4-isopropyl-3,4-dihydro-1,1′-biphenyl (103) (122The proposed mechanism for the synthesis of isopropyl biphenyl derivatives starts with yl (103) .1223.33.3.1124 A solution of biphenyl (2) and anhydrous tin(iv)chloride (SnCl4) in DCM was stirred, then lead tetraacetate Pd(OAc)4 was added in portion to produce 4-chloro-1,1′-biphenyl (107).125 Whereby, dichlorobiphenyl 108 was synthesized through passing chlorine gas (Cl2) to a solution of biphenyl (2) in glacial acetic acid (AcOH).126 On the other hands, biphenyl (2) was exposed to chlorine gas to obtain poly chlorinated biphenyl derivative 109 (127Polychlorinated biphenyls (PCBs) are a class of chlorinated organic compounds which were widely used in commercial and industrial applications. PCBs have many industrial applications including heat transfer, electrical, lubricants, and hydraulic equipment; as plastics, rubber products, coating, adhesive, capacitors, and plasticizers in paints; carbonless copy paper, and dyes.tive 109 .1273.3.2128,129 Stirring of biphenyl (2) with N-bromosuccinimide (NBS) in 2-methyltetrahydrofuran (2-MTHF) as solvent furnished mono bromobiphenyl 110.130 Whereas, the bromination of compound 2 by NBS in the presence of AgSbF6 and Trip-SMe in DCE furnished dibromo-biphenyl 111.131 Further, bromine gas (Br2) was mixed with biphenyl (2), cooled in an ice bath, and AlBr3 was added dropwise under the synthesis of HBr to afford PBBs 112, 113  have been employed as additives in polymers as well as flame retardants in the textile and electronic sectors.112, 113 .1293.3.3132 Lithiation of biphenyl (2) with n-butyllithium (n-BuLi) in the presence of tetra methyl ethylenediamine (TMEDA) led to formation of 2,2′-dilithiobiphenyl (114). Then, dilithiobiphenyl 114 reacted with iodine gas (I2) in THF to give 2,2′-diiodobiphenyl (115).133,134 On the other hand, a mixture of biphenyl (2), water, AcOH, concentrated sulfuric acid (H2SO4), I2, periodic acid (HIO4), and carbon tetrachloride (CCl4) was heated for 4 h to produce diiodobiphenyl 116.135 Furthermore, stirring of biphenyl (2) with dichloroiodoisocyanuric acid (DCICA) in CH3CN afforded 4-iodo-1,1′-biphenyl (117)  .1323.4136 The chloromethylation reaction of biphenyl (2) with formalin (HCHO) in the presence of HCl and FeCl3 produced 4-(chloromethyl)-1,1′-biphenyl (118) in 80% yield.137 Whereas, 4-(chloromethyl)-1,1′-biphenyl (118) was afforded in 78% yield through the treatment of biphenyl (2) with dimethoxymethane (CH2(OCH3)2) and chlorosulfonic acid (ClSO3H) in the presence of a catalytic amount of zinc iodide (ZnI2) in DCM  in DCM .1362(OCH3)2 with ClSO3H affords methyl chloromethyl ether. Then, ZnI2 promotes the production of the chloromethyl cation ([ClCH2]+). Finally, a chloromethylated derivative 118 is obtained by the electrophilic substitution reaction of biphenyl 2 with [ClCH2]+  was synthesized when liquid ammonia was added to Et2O containing biphenyl (2), followed by the addition of lithium metal.139 While, the treatment of biphenyl (2) with a noble metal catalyst  in the presence of Lewis acid (Hf(OTF)4) and octane (OCT) furnished 1,1′-bi(cyclohexane) (121).140 Whereas, nickel catalyzed hydrogenation of biphenyl (2) in the presence of isopropyl alcohol (i-PrOH) as a hydrogen donor afforded cyclohexylbenzene (122).141 Whereby, reduction of biphenyl 2 with a combination of samarium dibromide (SmBr2) and N-methylethanolamine (NMEA) as a chelating ligand furnished a mixture of 120 and 122 (142Reduction of biphenyl (2) with reducing lithium metal and ammonia afforded tetrahydro-1,1′-biphenyl 119. and 122 .1423.6o-dichlorobenzene (ODCB) with AlCl3, followed by adding a biphenyl solution of ODCB.143,144 In addition, biphenyl (2) was allowed to react with 2,4,6-trichloro-1,3,5-triazine (123) in the presence of hydrogen chloride (HCl) and AlCl3 in chlorobenzene yielded trisubstituted-6-chloro-1,3,5-triazine 124a–c -1,3,5-triazin-2-amine (124a) and 4,6-di-1,3,5-triazin-2-ol (124b) were synthesized by heating 4,6-dichloro-1,3,5-triazin derivatives 123 with a solution of e 124a–c .1453.7146 Luke et al.147 investigated the use of β-aryl-β-ketophosphonates as bone anabolic agent, thyroid receptor ligands, and tumor cell profiler inhibitors. Dimethyl(2-([biphenyl]-4-yl)-2-oxoethyl)phosphonate 126 was synthesized by stirring biphenyl (2) with trifluoroacetic anhydride (TFAA), which was then treated with dimethylphosphonoacetic acid (125) and phosphoric acid (H3PO4). Under purified nitrogen condition, a mixture of sodium nitrate (NaNO3) in trifluoroacetic acid (TFA), ACOH, and Ac2O was mixed with a solution of biphenyl (2) afforded acetate (127).146 Erbium(iii) triflate (Er(OTf)3) catalyzed Friedel–Crafts acylation of biphenyl (2) using acylating agent, such as benzoic acid (125) under microwave irradiation furnished -4-yl(phenyl)methanone (128).148 Furthermore, (3-amino-phenyl)-biphenyl-4-yl-methanone (129) was obtained by adding 3-aminobenzoic acid (125) and polyphosphoric acid (PPA) to biphenyl (2)  .1493.8et al.150 emphasized that several tetronic acid derivatives displayed a wide range of pharmacological activities, including acaricidal, insecticidal, HIV-I protease inhibitory, anti-inflammatory, antineoplastic, and cyclooxygenase inhibitory activity. Under conventional heating, synthesis of 1,1′-bipheny-4-hydroxyfuran-2(5H)-one 131 was achieved in excellent yield (51%) by reaction of biphenyl (2) and 3-diazofuran-2,4-dione 130. Gomberg–Bachmann–Hey reactions of benzendiazonium tetrafluoroborate derivatives 132 with biphenyl (2) in the presence of copper catalyst and cesium carbonate (Cs2CO3) in CH3CN afforded terphenyl derivatives 133.151 In the same context, heterogeneous gold(i)-catalyzed reaction of biphenyl (2) with 2-chlorophenyl-2-diazoacetate 134 afforded methyl 2-([biphenyl]-4-yl-2-chlorophenyl)acetate 135 (152Manchoju tate 135 .152i)-catalyzed mechanism of inactivated arenes is passed via sequence steps. Initially, for the synthesis of diazonium ion intermediate 135A, the cationic gold MCM-41–PPh2–AuOTf exchanges ligands with the diazo ester 134. Then, intermediate 135A collapses to produce the MCM-41-bound gold(i)carbene intermediate 135B, followed by combination between the nucleophilic arene 2 and the electrophilic gold(i)carbene intermediate 135B, the gold ionic intermediate 135C is produced. Last, intermediate 135C goes through 1,4-H shift and deauration to afford product 135 and MCM-41–PPh2–AuOTf complex is regenerated  with saccharin derivatives 136 in the presence of 1,10-phenanthroline (phen), Kives 137 .1533.103with SO3H-functionalized magnetic core/shell nano catalyst in DCM furnished 4-nitro-1,1′-biphenyl (138).154 While, treatment of a biphenyl with nitric acid (HNO3) in CCl4 solvent afforded 2-nitro-biphenyl 139  gave various oriented nitrated products 138 and 139 depending upon the nature of conditions and reagents applied on the reaction, as the nitration of biphenyl (2) was employed by refluxing NaNOenyl 139 .1553.11H-dibenzothiophen-5-ium-hexafluorophosphate(v) (141) was obtained in two steps  with AcOH and Ac2O in DCM, followed by adding sulfuric acid (H2SO4) dropwise. In the second stage, the intermediate product was neutralized by potassium hexafluorophosphate (KPF6) in water. Under the same conditions, compounds 143 and 145 were produced by reacting biphenyl (2) with thianthrene 5-oxide (142) and 2-chloro-9H-thioxanthen-9-one 10-oxide (144), respectively -5wo steps  startingectively .1563.12through stirring phloroglucinol (146) with AlCl3 in chlorobenzene (PhCl), followed by adding biphenyl . Biphenyl sulfonanilide derivatives 149 were produced in wide range yields (33–87%) by adding meta-chloroperbenzoic acid (mCPBA) to a stirred solution of sulfonamide derivatives 148, biphenyl 2, and iodobenzene (PhI) in a mixture of hexafluoroisopropanol (HFIP) and DCM  and a mixture of CH3CN and pyridine followed by adding piperidine afforded aminobiphenyl 150  as a nucleophile with biphenyl (2) as the arene coupling partner in the presence of a catalytic amount of mesityl acridinium salt, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), and a mixture of DCE and H2O afforded para and ortho aminobiphenyl products 150 and 151 respectively  in a solution of tetrabutylammonium tetrafluoroborate  synthesis involves the nucleophilic attack of pyridine on biphenyl (2), followed by one-electron oxidation to produce the intermediate ion 150A which is underwent aromatization process to yield drolysis .159160On the other hand, the proposed mechanism for the formation of aminobiphenyl 150 involves photoinduced electron transfer (PET) of biphenyl 2 in the presence of acridinium salt as a catalyst to an excited state photoredox radical (cat*) and biphenyl cation radical 150A, which reacted with amine derivatives to yield distonic cation radical 150B. Then, the deprotonation reaction of 150B furnished the radical intermediate 150C, which led to production of aminobiphenyl 150 by oxidative aromatization .1603.15161 reported the synthesis of biphenyl-4-yl-naphthalene 153via the treatment of bromo-biphenyl 2 with naphthalen-2-yl-boronic acid (152) in the presence of tetrabutylammonium bromide (TBABr), K3PO4, Pd(OAc)2 and Ni complex in a mixture of dioxane and water (Kashid and coworkersnd water .3.162pin274 in the presence of potassium t-butoxide (t-BuOK) and  (methoxy)iridium(i) dimer ([Ir(OMe)COD]2) catalyst in THF with dimethylbipyridyl (dmbpy) afforded tetrakis(Bpin)biphenyl 154.162 On the other hand, refluxing of biphenyl 2 with B2pin274 and a catalytic amount of N-heterocyclic carbene platinum(0) complex (IPr*Pt(dvtms)) gave two different oriented products namely, meta-(Bpin)biphenyl (155) and para-(Bpin)biphenyl (156) (163The borylation reaction of biphenyl (2) with Byl (156) .1633.17H-pyran-2,6-dione in the presence of 4-amino glucose functionalized silica propyl coated nickel ferrite nanoparticle (NiFe2O4@SiO2-propyl@glucosamine) catalyst and H2O were stirred at room temperature for 1 h afforded biphenyl amide derivative 158 (164Multicomponent reaction (MCRs) of biphenyl 2, 2-aminobenzoimidazole (157), and dihydro-2tive 158 .1643.18tert-butyl isocyanide mediated by photoactive electron acceptor donor complex (EDA) such as tris(4-bromophenyl)amine in the presence of Tf2O, DCE and basic additive (NaOAc) under blue-light irradiation produced biphenyl carbonitrile 159  with trimethylsilyl cyanide (TMSCN) in the presence of acridinium photocatalyst and phosphate buffer in O2-sparged CH3CN.166One-pot arene C–H cyanation reaction of biphenyl 2, phenoxathiine-10-oxide and rile 159 .165 Whervia interrupted Pummerer reactivity. The sulfonium salt 159A label's electron-deficient heteroaromatic system can form photoactive electron donor–acceptor (EDA) complexes with triaryl amine donors. Blue-light irradiation of the EDA complex causes single-electron transfer (SET) from the amine donor to salt 159A, which forms the radical cation of the electron donor and stimulates the synthesis of the aryl radical 159B. The radical trap 159B intercepting the aryl radical intermediate produces open shell species 159C, which are prone to oxidation to yield cations 159D, then the active electron-donor catalyst is regenerated. Finally, fragmentation of either tert-butyl isocyanide cation from 159D yields the required C–H functionalization products 159  undergoes oxidation using molecular oxygen to give phenylbenzonitrile 159. Finally, molecular oxygen or hydroperoxy radical oxidizes Mes-Ac radical to reproduce the catalyst  with biphenyl derivative 2 in trifluoromethanesulfonic anhydride (Tf2O) and DCM at ambient conditions furnished -4-yl(trifluoromethyl)sulfane derivatives 161 in range yields (65–82%)  .1672O and CF3SO2Na to afford an intermediate sulfonate sulfinate anhydride through self-disproportionation. Two possible routes lead to the final product. In path I, half of sulfonate sulfinate anhydride intermediate was oxidized to form TfO-SO2CF3 and another part is reduced to form CF3SOSO2CF3. Finally, the transfer of trifluoromethylthiolation from CF3SOSO2CF3 to biphenyl (2) to afford SCF3 product 161 sulfane 161 starts with the promotion of trifluoromethylthiolation using Tfduct 161 .1673.20N-Aryl azoles are a key motif in a number of biologically active compounds used in medicinal chemistry and crop protection science. The reaction of biphenyl (2) with parent pyrazole 162 in a mixture of HFIP and DCM,168 or in the presence of dipotassium peroxodisulfate (K2S2O8) and CH3CN under O2 atmosphere produced 4′-(1H-pyrazol-1-yl)--4-ylium (163).169 Whereby, Sen et al.170 reported oxidative azolation of unactivated biphenyl derivatives via using phenalenyl-based organic Lewis acid as powerful photooxidant in DCE to afford compound 163. While, under blue light irradiation, the photocatalytic oxidation of biphenyl 2 with azole 162 using DDQ catalyst and tert-butyl nitrite (TBN) yielded 1H-pyrazol-biphenyl-4-ylium 163 and 1--1H-pyrazole (164) (171le (164) .1713.21through two stages. First step involving a mixture of NBS, and 1-ethan-1-one (93) was refluxed in a mixture of petroleum ether (PET) and methanol for 2 h afforded 1--2-bromoethan-1-one (165). In the second stage, compound 165 was reacted with different phenyl carboxylic acid derivatives in the presence of K2CO3 in DMF at room temperature led to formation of 2--2-oxoethyl benzoate derivatives 166a. Biphenyl-4-yl-oxoethyl-pyridine carboxylate derivatives 166b were obtained by stirring of 1--2-bromoethan-1-one (165) with pyridine carboxylic acid derivatives in DMF containing anhydrous K2CO3  with dibromomethane afforded dibenzodioxepine 169. Furthermore, treatment of biphenyl-2,2′-diol (167) with 1,3-dibromopropane and K2CO3 in EtOH gave a mixture of 2′-(3-bromopropoxy)-[biphenyl]-2-ol 170a and bis(3-bromopropoxy)biphenyl 170b. In the same manner, a mixture of biphenyl-diol derivative 167 and methyl carbonochloridate in EtOH afforded biphenyl derivative 171. It is worthy to mention that o-alkylated biphenyl derivatives exhibit excellent scavenging of superoxide anion radical and β-glucuronidase inhibition activity (12Compounds containing biphenyl underwent electrophilic substitution to aromatic substrates. Dihydro-6activity .124OH) gave tricyclic ring of 4-biphenyl-1,4-dihydropyridine derivatives 173 and pentacyclic ring of [4-biphenyl]-1,7-dihydrodipyrazolopyridine derivatives 175, respectively. Then, oxidation of 173 and 175 with nitric acid (HNO3) afforded 4-(biphenyl-4-yl)-pyridine derivatives 174 and 4-biphenyl-dipyrazolopyridine derivatives 176, respectively which are used as anti-microbial resistance, antibacterial, antifungal and anticancer  with active methylene namely acetone derivatives or substituted pyrazole derivatives and ammonium hydroxide (NHticancer .1724) afforded biphenyl diol 178, followed by bromination reaction with phosphorus tribromide (PBr3) produced biphenyl dibromide 179. Then, compound 179 underwent cyclization reaction to produce azepine derivative 180. Finally, dealkylation of compound 180 furnished biphenyl diazepine derivative 181 -4-hydroperoxybutan-1-one (182), then coupling the product 182 with phenyl boronic acid (7)  and analgesic drug with less irritative effect on gastrointestinal tract . The synacid (7) .15l-valine methyl ester hydrochloride (184) with valeryl chloride in the presence of triethylamine (Et3N) in DCM to produce methyl N-pentanoyl-l-valinate (185). N-Protection of compound (185) with 1-bromo-4-(bromomethyl) benzene (186) in the presence of sodium hydride (NaH) in THF furnished methyl N-(4-bromobenzyl)-N-pentanoyl-d-valinate (187). Then, organozinc compound was synthesized via o-metalation of 5-phenyl-1-trityl-1H-tetrazole (188) with n-butyllithium (n-BuLi), followed by adding zinc chloride (ZnCl2). Negishi coupling reaction of organozinc chloride compound with aryl bromide 187 in the presence of palladium acetate (Pd(OAc)2) with 1,2,3,4,5-pentaphenyl-1′-(di-tert-butylphosphino)ferrocene (Q-phos) ligand in THF afforded N-pentanoyl-N-(2′-(1-trityl-1H-tetrazol-5-yl)--4-yl)-valinate (189). Finally, valsartan 190 was produced via hydrolysis of ester 189 with sodium hydroxide  receptor class and is used to treat cardiovascular disorders that are connected to hypertension. Synthesis of valsartan (190) began with coupling reaction of ydroxide .2053)4, 1,2-dimethoxyethane and Na2CO3 produced flurbiprofen (197). Flurbiprofen, a prostaglandin biosynthesis inhibitor, is extensively used as potent anti-inflammatory, antifungal, antipyretic, analgesic, in addition to, treatment Alzheimer's disease (AD)  197 has been achieved in subsequent steps. First step involving the reaction of 2,4-difluoronitrobenzene (191) with diethyl 2-methylmalonate (192) in the presence of NaH to afford diethyl 2-(3-fluoro-4-nitrophenyl)-2-methylmalonate (193), followed by catalytic hydrogenation of compound 193 to 4-amino-3-fluorophenyl-2-methylmalonate 194. After that, diazotization of the amino compound 194 into corresponding diazonium salt which then reacted with potassium iodide (KI) to produce diethyl 2-(3-fluoro-4-iodophenyl)-2-methylmalonate (195). Compound 195 underwent hydrolysis and decarboxylation reaction to yield 2-(3-fluoro-4-iodophenyl)propanoic acid (196). Finally, SMC reaction of 3-fluoro-4-iodophenylpropanoic acid 196 with phenylboronic acid in the presence of a catalytic amount of Pd(PPhase (AD) .206,2072O as a surfactant to afford 3-bromo-pyridin-3-yl)-2-methylbenzamide 200. Then, SMC reaction of compound 200 with (4-(trifluoromethoxy)phenyl)boronic acid (7) in the presence of Pd(OAc)2, PPh3, Pd, K3PO4 and THF  in the presence of coupling agent such as dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)pyridine (DMAP) and TPGS-750-M-H and THF .208through stirring a mixture of anisole (202), isoamyl nitrite (203), and 2,4-difluoroaniline (204) in the presence of cupper(i)salt. Demethylation reaction of difluorophenylanisole (205) with hydrogen iodide (HI) afforded 4--phenol (206). Kolbe–Schmitt phenol carboxylation of 206 with carbon dioxide (CO2) in the presence of K2CO3 afforded diflunisal (207). Whereby, diflunisal (207) has anti-inflammatory, anti-fever, and analgesic properties. In cases of osteoarthritis and rheumatoid arthritis, it is utilized to relief pain anisole (205) ief pain .209,210t-Bu afforded biphenylamide derivative 210. Then, cyclization reaction of compound 210 in the presence of TFA afforded imidazobiphenyl carbonitrile derivative 211. Alkylation reaction of compound 211 with dihaloalkane furnished spirocyclic compound 212.211 Finally, irbesartan (213) was synthesized via reaction of key intermediate 212 with sodium azide, tributyltin chloride (TBTC) and dimethoxycyclohexadiene 213. In pancreatic cancer, irbesartan overcomes gemcitabine resistance by decreasing iron metabolism and stemness via inhibition of the Hippo/YAP1/c-Jun axis.212 In addition, irbesartan lowers cholesterol via inhibiting the expression of the low-density lipoprotein receptor. Furthermore, irbesartan is used for the treatment of hypertension and type 2 diabetes (213Reaction of glycine methylester 208 with pentanoyl chloride in TEA produced methyl pentanoylglycinate (209). Compound 209 reacted with biphenylcarbonitrile derivative 16 in the presence of KOdiabetes .2136.214 Whereas, biphenyl biaryl pyrimidine derivatives (II) are utilized to treat AIDS and are expected to be anti-HIV drugs.215 5′-amino)-2-oxoethyl)amino)-2′-hydroxy--3,3′′-dicarboxylic acid (III) is developed to treat malignant tumours and as a transcription factor.216 3--2-hydroxyethoxy) phenol (IV) is a therapy for a disorder or disease accompanied by high activation of the NMDA receptor.217 4-(1-Substituted phenylvinyl)biphenyl derivatives (V) can be used for the manufacture of protein kinase, tubulin inhibitors and drugs for colon and breast cancer.218 2-[amino]nicotinic acid is used to treat psoriasis disease.219 (2E)-pyrimidine-3-yl](4-nitrophenyl)-prop-2-en-1-one, 2-(biphenyl)imidazopyrimidine-3-carbaldehyde and 6-pyrimidine-3-yl]-4-nitro pyrimidine-2(1H)-one exhibit promising antitumor activity by reducing the level of liver function enzyme near the normal level after damaged by CCl4.220 Whereby, trimethoxy--2-ol (VI) displays anti-inflammatory activity221 (Biphenyl sulfothiadiazole derivatives (I) are used in clinical immunotherapy of tumour patients and act as a PD1/PDL1 inhibitor.ivity221 .7.via metallic catalyzed reaction which covered the literature reported in the last decades based on this thrilling area of research. In this review several chemical reactions of biphenyl with different reagents have been discussed. The profound discussion of different captivating mechanisms associated with the role of numerous key catalysts and reagents through this review is believed to be beneficial for the synthetic community to apply these methods for practical purposes. Additionally, the efficacy of biphenyl compounds in medicinal chemistry and academic industry has been covered, and thereby will be helpful for future research. Although, obtaining these frameworks with particular substitution patterns is still an intimidating exercise. Atropisomerization phenomena of biphenyl derivatives also has taken into account Therefore, we believe that this review can be a guiding principle for synthetic and medicinal chemists working in this field.With the continuously increasing importance of biphenyl scaffolds as the structurally decisive scaffolds in bioactive natural products and pharmaceuticals drugs, formidable efforts have been carried out to summarize gradual developments of biphenyl compounds and their isosteres The authors confirm that this article content has no conflict of interest.2NCO2−H4N+HAmmonium carbamateADAlzheimer's diseaseATRAtrans retinoic acidAll-2O4@SiO2-propyl@glucosamineNiFe4-Amino glucose functionalized silica propyl coated nickel ferrite nanoparticleAeAlkali metals earth2Pd(dba)Bis(dibenzylideneacetone)palladium2(PPh3)2PdClBis-triphenylphosphine palladium dichloride2pin2BBis(pinacolato)diboronIMesH-imidazol-2-ylidene1,3-Bis-1,3-dihydro-2-6H4)OPPh2(2-BrC(2-Bromophenyl)diphenylphosphine oxide2CH2Cl2]i) dimer(methoxy)iridium(2CoClii) chlorideCobaltpyridineFtsZFilamenting temperature-sensitive mutant Z proteinHTSHigh-throughput screeningHFIP1,1,1,3,3,3-HexafluoroisopropanolLCDsLiquid crystal displays4LiAlHLithium aluminium hydrideMOFMetal–organic frameworks2-MTHF2-MethyltetrahydrofuranmCPBAmeta-Chloroperbenzoic acidMCRsMulticomponent reactionNBSN-BromosuccinimideNSAIDsNon-steroidal anti-inflammatory drugNMEAN-MethylethanolamineOLEDsOrganic light-emitting diodes2(COCl)Oxalyl chlorideOMIMsOrganic polymer of intrinsic microporosityODCBO-DichlorobenzenePd/CNSPalladium-catalyst on porous carbon nanospheresNa[Pd-NAS]Palladium grafted on natural asphalt sulfonatePCBsPolychlorinated biphenylsPBBsPolybrominated biphenylsPPAPolyphosphoric acidphen1,10-Phenanthroline6KPFPotassium hexafluorophosphate2]2[Cp*RhClPentamethylcyclopentadienyl rhodium dichloride dimer3PBrPhosphorus tribromideSMCSuzuki–Miyaura coupling reaction3SO2NaCFSodium trifluoromethanesulfinate6AgSbFSilver hexafluoroantimonate (V)2OAgSilver oxide2SmBrSamarium dibromideSETSingle-electron transfer3PPhTriphenylphosphineTEATriethylamine2(dba)3)(PdTris(dibenzylideneacetone)dipalladium(0)2SOClThionyl chloride3SnClPhTriphenyltin chloridetfpo-furylphosphineTris-4SnCliv)chlorideTin(3PBuTributyl phosphine3CHClTrichloromethanet-Boctert-Butyloxycarbonyl3)4Pd(PPhTetrakis(triphenylphosphine)palladiumTMEDATetra methyl ethylenediamineTFAATrifluoroacetic anhydride4NBF4BuTetrabutylammonium tetrafluoroborateTEMPO2,2,6,6-Tetramethylpiperidine-1-oxylTBABrTetrabutylammonium bromideTMSCNTrimethylsilyl cyanide2OTfTrifluoromethanesulfonic anhydrideTBNtert-Butyl nitriteTBTCTributyltin chloride