Investigation of the Efficacy of Epidermal Growth Factor Receptor (EGFR)-Tyrosine Kinase Inhibitor in Patients With EGFR Exon 21 L858R Point Mutation-Positive Non-small Cell Lung Cancer.

BACKGROUND: Treatment of advanced non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) has a higher response rate than with conventional chemotherapy in patients positive for EGFR mutations. However, the efficacy of EGFR-TKI therapy may be reduced in patients positive for the EGFR exon 21 L858R point mutation. OBJECTIVE: To determine the clinical characteristics of patients with EGFR exon 21 L858R point mutation-positive NSCLC who are non-responders to EGFR-TKI therapy and the factors that predict response to EGFR-TKI therapy. METHODS: Patients with NSCLC treated with EGFR-TKIs were evaluated for response after treatment, and those who responded were compared with those who did not respond. RESULTS: Of 31 patients, 21 (67.7%) responded to EGFR-TKI therapy (the response group). There were significantly more programmed death ligand 1 (PDL1)-negative patients in the response group than in the non-response group. A significantly higher number of patients in the PDL1-positive group developed interstitial lung disease (ILD) after EGFR-TKI therapy than those in the PDL1-negative group. CONCLUSION: EGFR-TKI therapy is likely to be non-responsive in PDL1-positive patients with EGFR exon 21 L858R point mutation-positive NSCLC. The PDL1-positive group is at a high risk of developing ILD.

Molecular Modification Enhances Xylose Uptake by the Sugar Transporter KM_SUT5 of Kluyveromyces marxianus.

This research cloned and expressed the sugar transporter gene KM_SUT5 from Kluyveromyces marxianus GX-UN120, which displayed remarkable sugar transportation capabilities, including pentose sugars. To investigate the impact of point mutations on xylose transport capacity, we selected four sites, predicted the suitable amino acid sites by molecular docking, and altered their codons to construct the corresponding mutants, Q74D, Y195K, S460H, and Q464F, respectively. Furthermore, we conducted site-directed truncation on six sites of KM_SUT5p. The molecular modification resulted in significant changes in mutant growth and the D-xylose transport rate. Specifically, the S460H mutant exhibited a higher growth rate and demonstrated excellent performance across 20 g L-1 xylose, achieving the highest xylose accumulation under xylose conditions (49.94 µmol h-1 gDCW-1, DCW mean dry cell weight). Notably, mutant delA554-, in which the transporter protein SUT5 is truncated at position delA554-, significantly increased growth rates in both D-xylose and D-glucose substrates. These findings offer valuable insights into potential modifications of other sugar transporters and contribute to a deeper understanding of the C-terminal function of sugar transporters.

Synchronous Double Primary Lung Adenocarcinomas With EGFR L858R Point Mutation and MET Exon 14 Skipping Mutation.

Various driver mutations and the corresponding molecular-targeted drugs have been detected and developed in non-small cell lung cancer. There were many cases in which surgical specimens had happened to find double primary cancers. However, to our knowledge, our case was the first report of synchronous double primary lung adenocarcinomas harboring epidermal growth factor receptor (EGFR) L858R and mesenchymal-to-epithelial transition (MET) exon 14 skipping mutations. A 75-year-old Japanese woman with chronic heart and renal failures was referred to our department because of a growing nodule in the right upper lung field on chest X-ray films. Chest computed tomography (CT) detected a nodule in the right S1 and another nodule in the left S1+2. Bronchoscopic biopsy diagnosed the right S1 nodule as moderately differentiated adenocarcinoma. Oncomine Dx Target Test Multi-CDx system of the right S1 adenocarcinoma detected EGFR L858R mutation. The 18F-fluorodeoxyglucose positron emission tomography/CT showed abnormal uptakes both in the right S1 and the left S1+2 nodules, and in the bilateral inferior paratracheal lymph nodes. We made a diagnosis of c-stage IIIA (cT1bN2M0) of adenocarcinoma in the right S1 and suspected another primary lung cancer in the left S1+2. Considering her general conditions, comorbidities and wishes, we started osimertinib. The right S1 cancer achieved partial response (PR), while the left S1+2 nodule and lymph nodes enlarged. Aspiration cytology from the left supraclavicular lymph node showed adenocarcinoma. The FoundationOne® Liquid CDx tumor profiling test detected not only EGFR L858R, but also MET exon 14 skipping mutation. We made a diagnosis of another primary adenocarcinoma from the left S1+2 nodule (cT1bN3M0, c-stage IIIB) with MET mutation, and changed osimertinib to capmatinib. Although the left S1+2 cancer achieved and maintained PR by capmatinib, the right S1 cancer increased, and several new metastases appeared. The subsequent switch from capmatinib to osimertinib could not control cancers. In this case, we tried to switch monotherapies from osimertinib to capmatinib for double primary adenocarcinomas harboring different two driver mutations, according to each cancer progression. The temporal and spatial heterogeneity reinforces the need for primary tissue biopsy if dual primaries are suspected. Temporally distinct liquid biopsies, not standard at present, may be considered.

Pseudoendocrine sarcoma: a rare new entity with unique radiologic and pathologic/molecular characteristics.

Pseudoendocrine sarcoma is a rare, recently described intermediate grade sarcoma of uncertain phenotype that most commonly affects the paraspinal location in older patients with a distinctive endocrine/paraganglioma-like morphology and unique CTNNB1 point mutation. While these tumors appear as epithelial or even benign endocrine tumors, these lack markers for such and are highlighted by nuclear expression of beta-catenin. This case is the first among the previously reported only twenty-five cases of this entity, including one original series and a few case reports, to correlate the radiologic imaging with the pathologic features. Furthermore, this case illustrates the oldest-to-date patient with this unique location as a palpable painful chest wall/paraspinal location, with new morphologic observations and, finally, this is only the second case to have this specific CTNNB1 hotspot point mutation for this rare entity.

Scarless Baculovirus Genome Editing Using Lambda-Red Recombineering in E. coli.

Baculoviruses are widely used for their potential as biological pesticide and as platform for the production of recombinant proteins and gene therapy vectors. The Baculovirus Expression Vector System (BEVS) is used for high level of expression of (multiple) proteins in insect cells. Baculovirus recombinants can be quickly constructed by transposition of the gene(s) of interest into a so-called bacmid, which is a baculovirus infectious clone maintained as single-copy, bacterial artificial chromosome in Escherichia coli. A two-step homologous recombineering technique using the lambda-red system in E. coli allows for scarless editing of the bacmid with PCR products based on sequence homology. In the first step, a selection cassette with 50 bp homology arms, typically generated by PCR, is inserted into the designated locus. In the second step, the selection cassette is removed based on a negative selection marker, such as SacB or rpsL. This lambda-red recombineering technique can be used for multiple gene editing purposes, including (large) deletions, insertions, and even single point mutations. Moreover, since there are no remnants of the editing process, successive modifications of the same bacmid are possible. This chapter provides detailed instructions to design and perform two-step homologous recombineering of baculovirus bacmid DNA in E. coli. We present two case studies demonstrating the utility of this technique for creating a deletion mutant of the chitinase and cathepsin genes and for introducing a single point mutation in the baculovirus gene gp41. This scarless genome editing approach can facilitate functional studies of baculovirus genes and improve the production of recombinant proteins using the BEVS.

Salmonella enteritidis acquires phage resistance through a point mutation in rfbD but loses some of its environmental adaptability.

Phage therapy holds promise as an alternative to antibiotics for combating multidrug-resistant bacteria. However, host bacteria can quickly produce progeny that are resistant to phage infection. In this study, we investigated the mechanisms of bacterial resistance to phage infection. We found that Rsm1, a mutant strain of Salmonella enteritidis (S. enteritidis) sm140, exhibited resistance to phage Psm140, which was originally capable of lysing its host at sm140. Whole genome sequencing analysis revealed a single nucleotide mutation at position 520 (C → T) in the rfbD gene of Rsm1, resulting in broken lipopolysaccharides (LPS), which is caused by the replacement of CAG coding glutamine with a stop codon TAG. The knockout of rfbD in the sm140ΔrfbD strain caused a subsequent loss of sensitivity toward phages. Furthermore, the reintroduction of rfbD in Rsm1 restored phage sensitivity. Moreover, polymerase chain reaction (PCR) amplification of rfbD in 25 resistant strains revealed a high percentage mutation rate of 64% within the rfbD locus. We assessed the fitness of four bacteria strains and found that the acquisition of phage resistance resulted in slower bacterial growth, faster sedimentation velocity, and increased environmental sensitivity (pH, temperature, and antibiotic sensitivity). In short, bacteria mutants lose some of their abilities while gaining resistance to phage infection, which may be a general survival strategy of bacteria against phages. This study is the first to report phage resistance caused by rfbD mutation, providing a new perspective for the research on phage therapy and drug-resistant mechanisms.

Production of Esters in Escherichia coli Using Citrate Synthase Variants.

Acetate esters comprise a wide range of products including fragrances and industrial solvents. Biosynthesis of esters offers a promising alternative to chemical synthesis because such routes use renewable carbohydrate resources and minimize the generation of waste. One biochemical method for ester formation relies on the ATF1 gene from Saccharomyces cerevisiae, which encodes alcohol-O-acyltransferase (AAT) which converts acetyl-CoA and an exogenously supplied alcohol into the ester. In this study, the formation of several acetate esters via AAT was examined in Escherichia coli chromosomally expressing citrate synthase variants, which create a metabolic bottleneck at acetyl-CoA. In shake flask cultures, variant strains generated more acetate esters than the strains expressing the wild-type citrate synthase. In a controlled bioreactor, E. coli GltA[A267T] generated 3.9 g propyl acetate in 13 h, corresponding to a yield of 0.155 g propyl acetate/g glucose, which is 18% greater than that obtained by the wild-type GltA control. These results demonstrate the ability of citrate synthase variants to redistribute carbon from central metabolism into acetyl-CoA-derived biochemicals.

Utilizing codon degeneracy in the design of donor DNA for CRISPR/Cas9-mediated gene editing to streamline the screening process for single amino acid mutations.

Chlamydomonas reinhardtii, a unicellular green alga, has been widely used as a model organism for studies of algal, plant and ciliary biology. The generation of targeted amino acid mutations is often necessary, and this can be achieved using CRISPR/Cas9 induced homology-directed repair to install genomic modifications from exogenous donor DNA. Due to the low gene editing efficiency, the technical challenge lies in identifying the mutant cells. Direct sequencing is not practical, and pre-screening is required. Here, we report a strategy for generating and screening for amino acid point mutations using the CRISPR/Cas9 gene editing system. The strategy is based on designing donor DNA using codon degeneracy, which enables the design of specific primers to facilitate mutant screening by PCR. An in vitro assembled RNP complex, along with a dsDNA donor and an antibiotic resistance marker, was electroporated into wild-type cells, followed by PCR screening. To demonstrate this principle, we have generated the E102K mutation in centrin and the K40R mutation in α-tubulin. The editing efficiencies at the target sites for Centrin, TUA1, TUA2 were 4, 24 and 8% respectively, based on PCR screening. More than 80% of the mutants with the expected size of PCR products were precisely edited, as revealed by DNA sequencing. Subsequently, the precision-edited mutants were biochemically verified. The introduction of codon degeneracy did not affect the gene expression of centrin and α-tubulins. Thus, this approach can be used to facilitate the identification of point mutations, especially in genes with low editing rates.

Genetically modified pigs with CD163 point mutation are resistant to HP-PRRSV infection.

Porcine reproductive and respiratory syndrome (PRRS) is a globally prevalent contagious disease caused by the positive-strand RNA PRRS virus (PRRSV), resulting in substantial economic losses in the swine industry. Modifying the CD163 SRCR5 domain, either through deletion or substitution, can eff1ectively confer resistance to PRRSV infection in pigs. However, large fragment modifications in pigs inevitably raise concerns about potential adverse effects on growth performance. Reducing the impact of genetic modifications on normal physiological functions is a promising direction for developing PRRSV-resistant pigs. In the current study, we identified a specific functional amino acid in CD163 that influences PRRSV proliferation. Viral infection experiments conducted on Marc145 and PK-15 CD163 cells illustrated that the mE535G or corresponding pE529G mutations markedly inhibited highly pathogenic PRRSV (HP-PRRSV) proliferation by preventing viral binding and entry. Furthermore, individual viral challenge tests revealed that pigs with the E529G mutation had viral loads two orders of magnitude lower than wild-type (WT) pigs, confirming effective resistance to HP-PRRSV. Examination of the physiological indicators and scavenger function of CD163 verified no significant differences between the WT and E529G pigs. These findings suggest that E529G pigs can be used for breeding PRRSV-resistant pigs, providing novel insights into controlling future PRRSV outbreaks.

Insecticide resistance and characteristics of mutations related to target site insensitivity of diamondback moths in Taiwan.

Diamondback moth (DBM, Plutella xylostella) is the most significant pest of cruciferous vegetables as they rapidly develop high-level resistance to many insecticides. Monitoring DBM susceptibility and target-site mutation frequency is essential for pest control. In this study, 10 insecticides were tested on 11 field populations. Frequencies of target-site mutations (including para, ace1, Rdl1, RyR1, and nAChRα6 genes) were estimated by pyrosequencing. Insecticides registered after 2007 for DBM control in Taiwan, i.e., spinetoram, chlorantraniliprole, chlorfenapyr, metaflumizone, and flubendiamide, showed >80% mortality toward several populations; Bacillus thurigiensis, emamectin benzoate, and chlorfluazuron showed medium to low efficacy in all populations; and tolfenpyrad and mevinphos were highly ineffective. Susceptibility to insecticides varied substantially among populations: eight out of nine populations were highly susceptible to spinetoram, but only one was susceptible to flubendiamide. Target-site mutations related to organophosphates, pyrethroids, fipronil, and diamides were detected in all populations, but there were few spinosad and spinetoram mutations. Our three-year field study demonstrated rapid efficacy loss for all insecticides tested, particularly for more toxic insecticides. Skipped-generation selection of a field DBM strain to emamectin benzoate, metaflumizone, chlorantraniliprole, and flubendiamide revealed that mortality rates dropped from 60 to 80% to <10% after 6 generations. Next-generation sequencing was performed to identify possible target gene mutations. A resistance management program that considers the instability of resistance to some chemicals and pertinent data on resistance mechanisms should be established. Identifying compounds to overcome high-frequency field DBM point mutations could be beneficial for pest control.

Pygo-F773W Mutation Reveals Novel Functions beyond Wnt Signaling in Drosophila.

Pygopus (Pygo) has been identified as a specific nuclear co-activator of the canonical Wingless (Wg)/Wnt signaling pathway in Drosophila melanogaster. Pygo proteins consist of two conserved domains: an N-terminal homologous domain (NHD) and a C-terminal plant homologous domain (PHD). The PHD's ability to bind to di- and trimethylated lysine 4 of histone H3 (H3K4me2/3) appears to be independent of Wnt signaling. There is ongoing debate regarding the significance of Pygo's histone-binding capacity. Drosophila Pygo orthologs have a tryptophan (W) > phenylalanine (F) substitution in their histone pocket-divider compared to vertebrates, leading to reduced histone affinity. In this research, we utilized CRISPR/Cas9 technology to introduce the Pygo-F773W point mutation in Drosophila, successfully establishing a viable homozygous Pygo mutant line for the first time. Adult mutant flies displayed noticeable abnormalities in reproduction, locomotion, heart function, and lifespan. RNA-seq and cluster analysis indicated that the mutation primarily affected pathways related to immunity, metabolism, and posttranslational modification in adult flies rather than the Wnt signaling pathway. Additionally, a reduction in H3K9 acetylation levels during the embryonic stage was observed in the mutant strains. These findings support the notion that Pygo plays a wider role in chromatin remodeling, with its involvement in Wnt signaling representing only a specific aspect of its chromatin-related functions.

Citrate synthase variants improve yield of acetyl-CoA derived 3-hydroxybutyrate in Escherichia coli.

BACKGROUND: The microbial chiral product (R)-3-hydroxybutyrate (3-HB) is a gateway to several industrial and medical compounds. Acetyl-CoA is the key precursor for 3-HB, and several native pathways compete with 3-HB production. The principal competing pathway in wild-type Escherichia coli for acetyl-CoA is mediated by citrate synthase (coded by gltA), which directs over 60% of the acetyl-CoA into the tricarboxylic acid cycle. Eliminating citrate synthase activity (deletion of gltA) prevents growth on glucose as the sole carbon source. In this study, an alternative approach is used to generate an increased yield of 3-HB: citrate synthase activity is reduced but not eliminated by targeted substitutions in the chromosomally expressed enzyme. RESULTS: Five E. coli GltA variants were examined for 3-HB production via heterologous overexpression of a thiolase (phaA) and NADPH-dependent acetoacetyl-CoA reductase (phaB) from Cupriavidus necator. In shake flask studies, four variants showed nearly 5-fold greater 3-HB yield compared to the wild-type, although pyruvate accumulated. Overexpression of either native thioesterases TesB or YciA eliminated pyruvate formation, but diverted acetyl-CoA towards acetate formation. Overexpression of pantothenate kinase similarly decreased pyruvate formation but did not improve 3-HB yield. Controlled batch studies at the 1.25 L scale demonstrated that the GltA[A267T] variant produced the greatest 3-HB titer of 4.9 g/L with a yield of 0.17 g/g. In a phosphate-starved repeated batch process, E. coli ldhA poxB pta-ackA gltA::gltA[A267T] generated 15.9 g/L 3-HB (effective concentration of 21.3 g/L with dilution) with yield of 0.16 g/g from glucose as the sole carbon source. CONCLUSIONS: This study demonstrates that GltA variants offer a means to affect the generation of acetyl-CoA derived products. This approach should benefit a wide range of acetyl-CoA derived biochemical products in E. coli and other microbes. Enhancing substrate affinity of the introduced pathway genes like thiolase towards acetyl-CoA will likely further increase the flux towards 3-HB while reducing pyruvate and acetate accumulation.

Revealing the Mechanism of NLRP3 Inflammatory Pathway Activation through K+ Efflux Induced by PLO via Signal Point Mutations.

Trueperella pyogenes is an important opportunistic pathogenic bacterium widely distributed in the environment. Pyolysin (PLO) is a primary virulence factor of T. pyogenes and capable of lysing many different cells. PLO is a member of the cholesterol-dependent cytolysin (CDC) family of which the primary structure only presents a low level of homology with other members from 31% to 45%. By deeply studying PLO, we can understand the overall pathogenic mechanism of CDC family proteins. This study established a mouse muscle tissue model infected with recombinant PLO (rPLO) and its single-point mutations, rPLO N139K and rPLO F240A, and explored its mechanism of causing inflammatory damage. The inflammatory injury abilities of rPLO N139K and rPLO F240A are significantly reduced compared to rPLO. This study elaborated on the inflammatory mechanism of PLO by examining its unit point mutations in detail. Our data also provide a theoretical basis and practical significance for future research on toxins and bacteria.

Increased terbinafine resistance among clinical genotypes of Trichophyton mentagrophytes/T. interdigitale species complex harboring squalene epoxidase gene mutations.

Terbinafine resistance has become epidemic as an emerging problem in treatment of dermatohpytosis. This could be attributed in part to a point mutation in the squalene epoxidase (SQLE) gene. In this study, point mutations in the SQLE gene were studied in T. rubrum and T. mentagrophytes/T. interdigitale species complex as two main causative agents of dermatophytosis. Antifungal susceptibility of clinical isolates of T. rubrum (n = 27) and T. mentagrophytes/T. interdigitale (n = 56) was assessed using the M38-3rd edition CLSI method. The SQLE gene and ITS region were sequenced for all the fungal strains, and the mutation sites and genotypes of the terbinafine-resistant strains were characterized. The results demonstrated that, in T. rubrum, the minimum inhibitory concentration of terbinafine (MIC50 and MIC90) was 0.03 µg/ml, and the geometric mean (G mean) concentration was 0.02. For the T. mentagrophytes complex, the MIC50 and MIC90 were 0.03 and 1.0 µg/ml, respectively, and the G mean concentration was 0.04 µg/ml. Four out of the five resistant strains were T. indotineae harboring the F397L and Q408L mutations, while the last one was T. mentagrophytes genotype VII, which harbors the F397L mutation. T. indotineae was the prominent causative agent of terbinafine resistance, with 80 % of the isolates, and T. mentagrophytes genotype VII was introduced as a new genotype in the terbinafine-resistant T. mentagrophytes complex. Our findings further substantiate the importance of antifungal susceptibility testing in selecting the choice of drug for effective treatment of dermatophytosis and highlight the importance of screening dermatophyte species for point mutations responsible for newly developed resistant strains to improve the current knowledge of overcoming infections caused by resistant species.

Induction of point and structural mutations in engineered yeast Saccharomyces cerevisiae improve carotenoid production.

ß-Carotene is an attractive compound and that its biotechnological production can be achieved by using engineered Saccharomyces cerevisiae. In a previous study, we developed a technique for the efficient establishment of diverse mutants through the introduction of point and structural mutations into the yeast genome. In this study, we aimed to improve ß-carotene production by applying this mutagenesis technique to S. cerevisiae strain that had been genetically engineered for ß-carotene production. Point and structural mutations were introduced into ß-carotene-producing engineered yeast. The resulting mutants showed higher ß-carotene production capacity than the parental strain. The top-performing mutant, HP100_74, produced 37.6 mg/L of ß-carotene, a value 1.9 times higher than that of the parental strain (20.1 mg/L). Gene expression analysis confirmed an increased expression of multiple genes in the glycolysis, mevalonate, and ß-carotene synthesis pathways. In contrast, expression of ERG9, which functions in the ergosterol pathway competing with ß-carotene production, was decreased in the mutant strain. The introduction of point and structural mutations represents a simple yet effective method for achieving mutagenesis in yeasts. This technique is expected to be widely applied in the future to produce chemicals via metabolic engineering of S. cerevisiae.

Investigation of genotype-phenotype and familial features of Turkish dystrophinopathy patients.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive allelic muscle diseases caused by dystrophin gene mutations. Eight hundred thirty-seven patients admitted between 1997 and 2022 were included in the study. Two hundred twenty patients were analyzed by multiplex PCR (mPCR) alone. Five hundred ninety-five patients were investigated by multiplex ligation-dependent probe amplification (MLPA), and 54 patients were examined by sequencing. Deletion was detected in 60% (132/220) of the cases in the mPCR group only and in 58.3% (347/595) of the cases with MLPA analysis. The rates of deletion and duplication were 87.7% and 12.3%, respectively, in the MLPA analysis. Single exon deletions were the most common mutation type. The introns 43-55 (81.8%) and exons 2-21 (13.1%) regions were detected as hot spots in deletions. It was determined that 89% of the mutations were suitable for exon skipping therapy. The reading frame rule did not hold in 7.6% of D/BMD cases (17/224). We detected twenty-five pathogenic/likely pathogenic variants in sequencing, five of which were novel variants. Nonsense mutation was the most common small mutation (44%). 21% of DMD patients were familial. We detected germline mosaicism in four families (4.3%) in the large rearrangement group and one gonosomal mosaicism in a family with a nonsense mutation. This is the largest study examining genotype and phenotype data in Turkish D/BMD families investigated by MLPA analysis. The reading frame hypothesis is not valid in all cases. Sharing the genotype and phenotype characteristics of these cases in the literature will shed light on the molecular structure of DMD and guide gene therapy research. In genetic counseling, carrier screening in the family and possible gonadal mosaicism should be emphasized.

Effects of RDL GABA Receptor Point Mutants on Susceptibility to Meta-Diamide and Isoxazoline Insecticides in Drosophila melanogaster.

Ionotropic γ-aminobutyric acid (GABA) receptors in insects, specifically those composed of the RDL (resistant to dieldrin) subunit, serve as important targets for commonly used synthetic insecticides. These insecticides belong to various chemical classes, such as phenylpyrazoles, cyclodienes, meta-diamides, and isoxazolines, with the latter two potentially binding to the transmembrane inter-subunit pocket. However, the specific amino acid residues that contribute to the high sensitivity of insect RDL receptors to these novel insecticides remain elusive. In this study, we investigated the susceptibility of seven distinct Drosophila melanogaster Rdl point mutants against four meta-diamide and isoxazoline insecticides: isocycloseram, fluxametamide, fluralaner, and broflanilide. Our findings indicate that, despite exhibiting increased sensitivity to fluralaner in vitro, the RdlI276C mutant showed resistance to isocycloseram and fluxametamide. Similarly, the double-points mutant RdlI276F+G279S also showed decreased sensitivity to the tested isoxazolines. On the other hand, the RdlG335M mutant displayed high levels of resistance to all tested insecticides. Molecular modeling and docking simulations further supported these findings, highlighting similar binding poses for these insecticides. In summary, our research provides robust in vivo evidence supporting the idea that the inter-subunit amino acids within transmembrane M1 and M3 domains form the binding site crucial for meta-diamide and isoxazoline insecticide interactions. This study highlights the complex interplay between mutations and insecticide susceptibility, paving the way for more targeted pest control strategies.

Recent advances in enzyme-free and enzyme-mediated single-nucleotide variation assay in vitro.

Single-nucleotide variants (SNVs) are the most common type variation of sequence alterations at a specific location in the genome, thus involving significant clinical and biological information. The assay of SNVs has engaged great awareness, because many genome-wide association studies demonstrated that SNVs are highly associated with serious human diseases. Moreover, the investigation of SNV expression levels in single cells are capable of visualizing genetic information and revealing the complexity and heterogeneity of single-nucleotide mutation-related diseases. Thus, developing SNV assay approaches in vitro, particularly in single cells, is becoming increasingly in demand. In this review, we summarized recent progress in the enzyme-free and enzyme-mediated strategies enabling SNV assay transition from sensing interface to the test tube and single cells, which will potentially delve deeper into the knowledge of SNV functions and disease associations, as well as discovering new pathways to diagnose and treat diseases based on individual genetic profiles. The leap of SNV assay achievements will motivate observation and measurement genetic variations in single cells, even within living organisms, delve into the knowledge of SNV functions and disease associations, as well as open up entirely new avenues in the diagnosis and treatment of diseases based on individual genetic profiles.

Genotyping Zebrafish Point Mutant by Allele-Specific Blocking PCR.

Genotyping zebrafish carrying wild-type, heterozygous, or homozygous copies of a mutant allele is often required for investigating gene specific functions, and is routinely performed to differentiate point mutants. In this study, we describe a modified allele-specific PCR method using an additional blocking primer to promote target sequence amplification while suppressing sequences with single mismatch. Using the tp53m214k point mutant as an example, we show that wild-type, heterozygous, and homozygous zebrafish can be easily distinguished using this simple PCR method, which could be widely adapted for genotyping zebrafish with point mutations or small nucleotide insertions/deletions.

Cancer-associated point mutations within the extracellular domain of PTPRD affect protein stability and HSPG interaction.

PTPRD, a well-established tumor suppressor gene, encodes the protein tyrosine phosphatase-type D. This protein consists of three immunoglobulin-like (Ig) domains, four to eight fibronectin type 3 (FN) domains, a single transmembrane segment, and two cytoplasmic tandem tyrosine phosphatase domains. PTPRD is known to harbor various cancer-associated point mutations. While it is assumed that PTPRD regulates cellular functions as a tumor suppressor through the tyrosine phosphatase activity in the intracellular region, the function of its extracellular domain (ECD) in cancer is not well understood. In this study, we systematically examined the impact of 92 cancer-associated point mutations within the ECD. We found that 69.6% (64 out of 92) of these mutations suppressed total protein expression and/or plasma membrane localization. Notably, almost all mutations (20 out of 21) within the region between the last FN domain and transmembrane segment affected protein expression and/or localization, highlighting the importance of this region for protein stability. We further found that some mutations within the Ig domains adjacent to the glycosaminoglycan-binding pocket enhanced PTPRD's binding ability to heparan sulfate proteoglycans (HSPGs). This interaction is proposed to suppress phosphatase activity. Our findings therefore suggest that HSPG-mediated attenuation of phosphatase activity may be involved in tumorigenic processes through PTPRD dysregulation.