C and G are frequently mutated into T and A in coding regions of human genes.

Nucleotide mutations in human genes have long been a hot subject for study because some of them may lead to severe human diseases. Understanding the general mutational process and evolutionary trend of human genes could help answer such questions as why certain diseases occur and what challenges we face in protecting human health. In this study, we conducted statistics on 89,895 single-nucleotide variations identified in coding regions of 18,339 human genes. The results show that C and G are frequently mutated into T and A in human genes. C/G (C or G)-to-T/A mutations lead to reduction of hydrogen bonds in double-stranded DNA because C-G and T-A base pairs are maintained by three and two hydrogen bonds respectively. C-to-T and G-to-A mutations occur predominantly in human genes because they not only reduce hydrogen bonds but also belong to transition mutation. Reduction of hydrogen bonds could reduce energy consumption not only in separating double strands of mutated DNA for transcription and replication but also in disrupting stem-loop structure of mutated mRNA for translation. It is thus considered that to reduce hydrogen bonds (and thus to reduce energy consumption in gene expression) is one of the driving forces for nucleotide mutation. Moreover, codon mutation is positively correlated to its content, suggesting that most mutations are not targeted on changing any specific codons (amino acids) but are merely for reducing hydrogen bonds. Our study provides an example of utilizing single-nucleotide variation data to infer evolutionary trend of human genes, which can be referenced to conduct similar studies in other organisms.

Feasibility and safety of laparoscopic radical cystectomy for male octogenarians with muscle-invasive bladder cancer.

This study was designed to evaluate the safety and feasibility of laparoscopic radical cystectomy (LRC) for male octogenarian patients with muscle-invasive bladder cancer (MIBC). Briefly, a total of 57 male octogenarian patients (A group) with bladder carcinoma were enrolled and underwent LRC and intracorporeal pelvic lymph node dissection with bilateral cutaneous ureterostomy from May 2016 to December 2022. Besides, 63 male patients (age < 80 years old) with bladder carcinoma undergoing LRC and 17 octogenarian male patients with bladder carcinoma undergoing open radical cystectomy (ORC) were enrolled in B and C groups as control. All perioperative clinical materials and outcomes of long-term follow-up, and complication were collected. The specific results were shown as follows. Compared with C group, the operation time and resected lymph node in A group was increased, and the estimated blood loss, the number of transfusion needed, duration of pelvic drainage and hospital stay after surgery was decreased. The death rate and ileus complication rate were higher in A group (12 cases) than in C group (15 cases). The cases of ureteral stricture in A group (13 cases) was decreased compared with that in C group. Overall, LRC and bilateral cutaneous ureterostomy are safe, feasible and better choices for the treatment of male octogenarian patients with MIBC. The octogenarian receiving cutaneous ureterostomy heals slowly and exists certain incomplete intestinal obstruction after surgery.

Characterization and Engineering of Two Highly Paralogous Sesquiterpene Synthases Reveal a Regioselective Reprotonation Switch.

Sesquiterpene synthases (STPSs) catalyze carbocation-driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation-reprotonation process is widely used in STPSs to promote structural diversity, largely attributable to the distinct regio/stereoselective reprotonations. However, the molecular basis for reprotonation regioselectivity remains largely understudied. Herein, we analyzed two highly paralogous STPSs, Artabotrys hexapetalus (-)-cyperene synthase (AhCS) and ishwarane synthase (AhIS), which catalyze reactions that are distinct from the regioselective protonation of germacrene A (GA), resulting in distinct skeletons of 5/5/6 tricyclic (-)-cyperene and 6/6/5/3 tetracyclic ishwarane, respectively. Isotopic labeling experiments demonstrated that these protonations occur at C3 and C6 of GA in AhCS and AhIS, respectively. The cryo-electron microscopy-derived AhCS complex structure provided the structural basis for identifying different key active site residues that may govern their functional disparity. The structure-guided mutagenesis of these residues resulted in successful functional interconversion between AhCS and AhIS, thus targeting the three active site residues [L311-S419-C458]/[M311-V419-A458] that may act as a C3/C6 reprotonation switch for GA. These findings facilitate the rational design or directed evolution of STPSs with structurally diverse skeletons.

MBD5 regulates NMDA receptor expression and seizures by inhibiting Stat1 transcription.

Epilepsy is considered to result from an imbalance between excitation and inhibition of the central nervous system. Pathogenic mutations in the methyl-CpG binding domain protein 5 gene (MBD5) are known to cause epilepsy. However, the function and mechanism of MBD5 in epilepsy remain elusive. Here, we found that MBD5 was mainly localized in the pyramidal cells and granular cells of mouse hippocampus, and its expression was increased in the brain tissues of mouse models of epilepsy. Exogenous overexpression of MBD5 inhibited the transcription of the signal transducer and activator of transcription 1 gene (Stat1), resulting in increased expression of N-methyl-d-aspartate receptor (NMDAR) subunit 1 (GluN1), 2A (GluN2A) and 2B (GluN2B), leading to aggravation of the epileptic behaviour phenotype in mice. The epileptic behavioural phenotype was alleviated by overexpression of STAT1 which reduced the expression of NMDARs, and by the NMDAR antagonist memantine. These results indicate that MBD5 accumulation affects seizures through STAT1-mediated inhibition of NMDAR expression in mice. Collectively, our findings suggest that the MBD5-STAT1-NMDAR pathway may be a new pathway that regulates the epileptic behavioural phenotype and may represent a new treatment target.

Functional and phylogenetic relationships link predators to plant diversity via trophic and non-trophic pathways.

Human-induced biodiversity loss negatively affects ecosystem function, but the interactive effects of biodiversity change across trophic levels remain insufficiently understood. We sampled arboreal spiders and lepidopteran larvae across seasons in 2 years in a subtropical tree diversity experiment, and then disentangled the links between tree diversity and arthropod predator diversity by deconstructing the pathways among multiple components of diversity (taxonomic, phylogenetic and functional) with structural equation models. We found that herbivores were major mediators of plant species richness effects on abundance, species richness, functional and phylogenetic diversity of predators, while phylogenetic, functional and structural diversity of trees were also important mediators of this process. However, the strength and direction differed between functional, structural and phylogenetic diversity effects, indicating different underlying mechanisms for predator community assembly. Abundance and multiple diversity components of predators were consistently affected by tree functional diversity, indicating that the variation in structure and environment caused by plant functional composition might play key roles in predator community assembly. Our study highlights the importance of an integrated approach based on multiple biodiversity components in understanding the consequences of biodiversity loss in multitrophic communities.

Antipsychotic drugs induce vascular defects in hematopoietic organs.

Antipsychotic agents are clinically utilized to treat schizophrenia and other mental disorders. These drugs induce neurological and metabolic side effects, but their influence on blood vessels remains largely unknown. Here, we show that haloperidol, one of the most frequently prescribed antipsychotic agents, induces vascular defects in bone marrow. Acute haloperidol treatment results in vascular dilation that is specific to hematopoietic organs. This vessel dilation is associated with disruption of hematopoiesis and hematopoietic stem/progenitor cells (HSPCs), both of which are reversible after haloperidol withdrawal. Mechanistically, haloperidol treatment blocked the secretion of vascular endothelial growth factor A (VEGF-A) from HSPCs. Genetic blockade of VEGF-A secretion from hematopoietic cells or inhibition of VEGFR2 in endothelial cells result in similar vessel dilation in bone marrow during regeneration after irradiation and transplantation. Conversely, VEGF-A gain of function rescues the bone marrow vascular defects induced by haloperidol treatment and irradiation. Our work reveals an unknown effect of antipsychotic agents on the vasculature and hematopoiesis with potential implications for drug application in clinic.

Tree dissimilarity determines multi-dimensional beta-diversity of herbivores and carnivores via bottom-up effects.

Global biodiversity decline and its cascading effects through trophic interactions pose a severe threat to human society. Establishing the impacts of biodiversity decline requires a more thorough understanding of multi-trophic interactions and, more specifically, the effects that loss of diversity in primary producers has on multi-trophic community assembly. Within a synthetic conceptual framework for multi-trophic beta-diversity, we tested a series of hypotheses on neutral and niche-based bottom-up processes in assembling herbivore and carnivore communities in a subtropical forest using linear models, hieratical variance partitioning based on linear mixed-effects models (LMMs) and simulation. We found that the observed taxonomic, phylogenetic and functional beta-diversity of both herbivorous caterpillars and carnivorous spiders were significantly and positively related to tree dissimilarity. Linear models and variance partitioning for LMMs jointly suggested that as a result of bottom-up effects, producer dissimilarities were predominant in structuring consumer dissimilarity, the strength of which highly depended on the trophic dependencies on producers, the diversity facet examined, and data quality. Importantly, linear models for standardized beta-diversities against producer dissimilarities implied a transition between niche-based processes such as environmental filtering and competitive exclusion, which supports the role of bottom-up effect in determining consumer community assembly. These findings enrich our mechanistic understanding of the 'Diversity Begets Diversity' hypothesis and the complexity of higher-trophic community assembly, which is fundamental for sustainable biodiversity conservation and ecosystem management.

Growth, development, and host preference of Histiostoma feroniarum (Acaridida: Histiostomatidae): effects of temperature and types of mushroom cultivar.

One of the most common harmful mites in edible fungi is Histiostoma feroniarum Dufour (Acaridida: Histiostomatidae), a fungivorous astigmatid mite that feeds on hyphae and fruiting bodies, thereby transmitting pathogens. This study examined the effects of seven constant temperatures and 10 types of mushrooms on the growth and development of H. feroniarum, as well as its host preference. Developmental time for the total immature stages was significantly affected by the type of mushroom species, ranging from 4.3 ± 0.4 days (reared on Pleurotus eryngii var. tuoliensis Mou at 28°C) to 17.1 ± 2.3 days (reared on Auricularia polytricha Sacc. at 19°C). The temperature was a major factor in the formation of facultative heteromorphic deutonymphs (hypopi). The mite entered the hypopus stage when the temperature dropped to 16°C or rose above 31°C. The growth and development of this mite were significantly influenced by the type of species and variety of mushrooms. Moreover, the fungivorous astigmatid mite preferred to feed on the 'Wuxiang No. 1' strain of Lentinula edodes (Berk.) Pegler and the 'Gaowenxiu' strain of P. pulmonarius (Fr.) Quél., with a shorter development period compared with that of feeding on other strains. These results therefore quantify the effect of host type and temperature on fungivorous astigmatid mite growth and development rates, and provide a reference for applying mushroom cultivar resistance to biological pest control.

Enzymatic synthesis of anhydroicaritin, baohuoside and icariin.

Anhydroicaritin (1a), baohuoside (1b) and icariin (1c) were recognized as major pharmacologically active ingredients of Epimedium plants. Their primary means of acquisition were chemical isolation from plants. However, it suffers from low yield, environmental pollution and shortage of plants. Herein, to remedy these problems, biosynthesis was explored to obtain the three active ingredients. Fortunately, with SfFPT as 8-prenyltransferase, EpPF3RT and Ep7GT as glycosyltransferases, kaempferide (1) was transferred to 1a, 1b and 1c enzymatically. Thus, we report the details of this method. This approach represents a promising environmental friendly alternative for the production of these compounds from an abundant analogue.

CD38+ B cells affect immunotherapy for allergic rhinitis.

BACKGROUND: Allergen-specific immunotherapy (AIT) is the mainstay in the treatment of allergic diseases, but the therapeutic effects of AIT need to be improved. CD38+ B cells are an immune cell fraction involved in the pathogenesis of allergic diseases as well as in immune regulation. OBJECTIVE: We sought to elucidate the role of antigen-specific CD38+ B cells in AIT. METHODS: An analysis was carried out on AIT results of 48 patients with perennial allergic rhinitis (AR), among which peripheral blood immune cells were analyzed by flow cytometry; serum cytokine levels were determined by ELISA. An AR murine model was developed to test the role of CD38+ B cells in AIT. RESULTS: A fraction of antigen-specific CD38+ B cell was detected in AR patients. CD38+ B-cell frequency was negatively correlated with the therapeutic effects of AIT. A negative correlation was detected between the CD38+ B-cell frequency and regulatory T-cell frequency in AR patients treated with AIT. Exposure to specific antigens induced CD38+ B cells to produce IL-6, that converted Treg cells to TH17 cells. Coadministration of anti-CD38 antibody significantly promoted the therapeutic effects of AIT. CONCLUSIONS: Antigen-specific CD38+ B cells compromise AIT effects by producing IL-6 to convert regulatory T cells to TH17 cells. Inhibition of CD38+ B cells promotes the effects of AIT.

Propionic acid regulates immune tolerant properties in B Cells.

Interleukin 10 (IL-10)-producing B cells (B10 cells) are a canonical cell fraction for regulating other activities of immune cells. Posttranscriptional modification of IL-10 in B10 cells is not yet fully understood. Short-chain fatty acids play an important role to regulate the functions of immune cells. This study aims to clarify the role of propionic acid (PA), a short-chain fatty acid, in regulating the expression of IL-10 in B10 cells. Blood samples were collected from patients with food allergy (FA) and healthy subjects. Serum and cellular components were prepared with the samples, and analysed by enzyme-linked immunosorbent assay and flow cytometry, respectively. The results showed that serum PA levels were lower in FA patients. PA concentrations were negatively correlated with serum cytokine Th2 concentrations, specific IgE concentrations in serum and skin prick test results. The peripheral frequency of B10 cells and the production of IL-10 in B cells were also associated with serum PA concentrations. Activation of B cells by CpG induced the production of IL-10 and tristetretrprolin (TTP), in which TTP caused the spontaneous decay of IL-10 mRNA. PA was necessary to stabilize the IL-10 mRNA in B cells by inducing the production of granzyme B, which resulted in the degradation of the IL-10 mRNA. Administration of PA attenuated FA response in mice by maintaining homeostasis of B10 cells. In conclusion, PA is needed to stabilize the expression of IL-10 in B10 cells. PA administration can mitigate experimental FA by maintaining B10 cell functions.

SKI knockdown suppresses apoptosis and extracellular matrix degradation of nucleus pulposus cells via inhibition of the Wnt/ß-catenin pathway and ameliorates disc degeneration.

This study aimed to determine the effects of SKI on interleukin (IL)-1ß-induced apoptosis of nucleus pulposus (NP) cells, intervertebral disc degeneration (IDD), and the Wnt signaling pathway. NP tissue specimens of different Pfirrmann grades (II-V) were collected from patients with different grades of IDD. Real-time polymerase chain reaction and western blotting were used to compare SKI mRNA and protein expression in NP tissues from patients. Using the IL-1ß-induced IDD model, NP cells were infected with lentivirus-coated si-SKI to downregulate the expression of SKI and treated with LiCl to evaluate the involvement of the Wnt/ß-catenin signaling pathway. Western blotting, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to detect NP cell apoptosis, extracellular matrix (ECM) metabolism, and related protein expression changes in the Wnt/ß-catenin signaling pathway. To investigate the role of SKI in vivo, a rat IDD model was established by needle puncture of the intervertebral disc. Rats were injected with lentivirus-coated si-SKI and evaluated by magnetic resonance imaging (MRI), and hematoxylin and eosin (HE) and safranin O staining. SKI expression positively correlated with the severity of human IDD. In the IL-1ß-induced NP cell degeneration model, SKI expression increased significantly and reached a peak at 24 h. SKI knockdown protected against IL-1ß-induced NP cell apoptosis and ECM degradation. LiCl treatment reversed the protective effects of si-SKI on NP cells. Furthermore, lentivirus-coated si-SKI injection partially reversed the NP tissue damage in the IDD model in vivo. SKI knockdown reduced NP cell apoptosis and ECM degradation by inhibiting the Wnt/ß-catenin signaling pathway, ultimately protecting against IDD. Therefore, SKI may be an effective target for IDD treatment.

Human activities have opposing effects on distributions of narrow-ranged and widespread plant species in China.

Human activities have shaped large-scale distributions of many species, driving both range contractions and expansions. Species differ naturally in range size, with small-range species concentrated in particular geographic areas and potentially deviating ecologically from widespread species. Hence, species' responses to human activities may be influenced by their geographic range sizes, but if and how this happens are poorly understood. Here, we use a comprehensive distribution database and species distribution modeling to examine if and how human activities have affected the extent to which 9,701 vascular plants fill their climatic potential ranges in China. We find that narrow-ranged species have lower range filling and widespread species have higher range filling in the human-dominated southeastern part of China, compared with their counterparts distributed in the less human-influenced northwestern part. Variations in range filling across species and space are strongly associated with indicators of human activities (human population density, human footprint, and proportion of cropland) even after controlling for alternative drivers. Importantly, narrow-ranged and widespread species show negative and positive range-filling relationships to these human indicators, respectively. Our results illustrate that floras risk biotic homogenization as a consequence of anthropogenic activities, with narrow-ranged species becoming replaced by widespread species. Because narrow-ranged species are more numerous than widespread species in nature, negative impacts of human activities will be prevalent. Our findings highlight the importance of establishing more protected areas and zones of reduced human activities to safeguard the rich flora of China.

Coupled liquid biopsy and bioinformatics for pancreatic cancer early detection and precision prognostication.

Early detection and diagnosis are the key to successful clinical management of pancreatic cancer and improve the patient outcome. However, due to the absence of early symptoms and the aggressiveness of pancreatic cancer, its 5-year survival rate remains below 5 %. Compared to tissue samples, liquid biopsies are of particular interest in clinical settings with respect to minimal invasiveness, repeated sampling, complete representation of the entire or multi-site tumor bulks. The potential of liquid biopsies in pancreatic cancer has been demonstrated by many studies which prove that liquid biopsies are able to detect early emergency of pancreatic cancer cells, residual disease, and recurrence. More interestingly, they show potential to delineate the heterogeneity, spatial and temporal, of pancreatic cancer. However, the performance of liquid biopsies for the diagnosis varies largely across different studies depending of the technique employed and also the type and stage of the tumor. One approach to improve the detect performance of liquid biopsies is to intensively inspect circulome and to define integrated biomarkers which simultaneously profile circulating tumor cells and DNA, extracellular vesicles, and circulating DNA, or cell free DNA and proteins. Moreover, the diagnostic validity and accuracy of liquid biopsies still need to be comprehensively demonstrated and validated.

Targeted therapy for intervertebral disc degeneration: inhibiting apoptosis is a promising treatment strategy.

Intervertebral disc (IVD) degeneration (IDD) is a multifactorial pathological process associated with low back pain (LBP). The pathogenesis is complicated, and the main pathological changes are IVD cell apoptosis and extracellular matrix (ECM) degradation. Apoptotic cell loss leads to ECM degradation, which plays an essential role in IDD pathogenesis. Apoptosis regulation may be a potential attractive therapeutic strategy for IDD. Previous studies have shown that IVD cell apoptosis is mainly induced by the death receptor pathway, mitochondrial pathway, and endoplasmic reticulum stress (ERS) pathway. This article mainly summarizes the factors that induce IDD and apoptosis, the relationship between the three apoptotic pathways and IDD, and potential therapeutic strategies. Preliminary animal and cell experiments show that targeting apoptotic pathway genes or drug inhibition can effectively inhibit IVD cell apoptosis and slow IDD progression. Targeted apoptotic pathway inhibition may be an effective strategy to alleviate IDD at the gene level. This manuscript provides new insights and ideas for IDD therapy.

Comparison between zero-profile and cage plate devices in the treatment of single-level cervical spondylopathy.

OBJECTIVE: To compare the new zero-profile (Zero-P) fusion cage with regular cage and plate (CP) in the treatment of anterior cervical single-level cervical degenerative diseases. METHODS: Patients operated from January 2013 to August 2018 were enrolled. They were divided into the Zero-P group (n = 74 cases) and CP groups (n = 116 cases). Follow-up assessment was at 1, 3, 12, and 24 months after surgery, including the incidence of dysphagia, visual analogue scale (VAS) score, Japanese orthopaedic association (JOA)score, C2-C7 Cobb angle, intervertebral disc height (IDH) and adjacent joint degeneration. RESULTS: The operation time and blood loss of patients in Zero-P group were less than those in the CP group, and there was no difference in hospitalization time. All 190 patients were followed up for 24 to 72 months, with an average of 35.29 months. In terms of clinical outcomes, vas and JOA scores of the two groups were significantly improved at one month and the last follow-up. The incidence of dysphagia in the Zero-P group was lower than that in the CP group. On radiological effects, Cobb angle and IDH showed significant correction in both groups, but the degeneration rate of adjacent joints in the Zero-P group was lower than the CP group. CONCLUSIONS: In ACDF, the clinical and radiological results of Zero-P and CP devices are satisfactory, but Zero-P cage may be superior in operation time, blood loss, the incidence of dysphagia and adjacent joint degeneration.

Phylogenetic analysis of achaete-scute complex genes in metazoans.

Achaete-scute complex (ASC) genes play essential roles in regulating neurogenesis of metazoans. Various metazoan species have greatly different numbers of genes in ASCa, ASCb and ASCc families. To explore evolutionary mechanisms of metazoan ASC genes, Blast (basic local alignment search tool) searches and phylogenetic analyses were conducted to identify ASC genes in metazoan species and to infer phylogenetic relationship between various ASC genes. As a result, 2784 ASC genes were identified in 804 metazoan species. The phylogenetic tree constructed using 1237 unique bHLH motifs shows that metazoan ASCa, ASCb and ASCc families contain six (a1-a6), five (b1-b5) and three (c1-c3) bHLH genes, respectively. Further phylogenetic analyses suggest that ASC genes in metazoans are derived from a primitive c gene, those in insects are derived from c2 gene, and those in chordates are derived from a2 and a3 genes. Data of gene linkage demonstrate that insect a6 is derived from a4 but not from a5, and chordate a2 is ancestral to b5 only, whilst a3 is ancestral to both b3 and b5. It is concluded that current ASC gene families in metazoans were established through a series of sub- and/or neo-functionalization to duplicated ancestral ASC gene(s). These results provide good references for exploring evolutionary mechanisms of other bHLH genes in metazoans. Besides, gene subtyping is considered as an efficient method for evolutionary studies on closely related homologous genes.

Ursolic acid potentiated oxaliplatin to induce apoptosis in colorectal cancer RKO cells.

Ursolic acid (UA) is found in multiple anticancer herbs and has shown anticancer effects in colorectal cancer (CRC) cells. The present study aimed to observe the effects of a combination of UA and oxaliplatin (Oxa), a frequently used chemotherapeutic drug in CRC, on human CRC RKO cells. The results showed that UA and Oxa synergistically inhibited the proliferation of RKO cells. A combination of UA and Oxa induced apoptosis in RKO cells and increased the activities of caspase-3, caspase-8, and caspase-9. Z-VAD-FMK, a caspase inhibitor, significantly antagonized UA- and Oxa-activated caspase-3, caspase-8, and caspase-9 and induced apoptosis. In addition, UA and Oxa downregulated the expression of X-linked inhibitor of apoptosis (XIAP) and Survivin in RKO cells. These observations suggested that a combination of UA and Oxa elicited synergistically anticancer effects in RKO cells and provided new evidence for potential application of UA and Oxa for CRC treatment.

Impact of the updated 2018 ASCO/CAP guidelines on HER2 FISH testing in invasive breast cancer: a retrospective study of HER2 fish results of 2233 cases.

OBJECTIVES: Human epidermal growth factor receptor 2 (HER2, ERBB2) is a valuable prognostic and predictive biomarker in breast cancer. Accurate assessment of HER2 status is essential in selecting the patients with invasive breast cancer who will likely response to HER2-targeted therapies. Some major modifications in the diagnostic recommendation for fluorescence in situ hybridization (FISH) have been made in the updated 2018 American Society of Clinical Oncology (ASCO)/College of American Pathologist (CAP) guideline. According to the revised guideline, concomitant IHC assays are required to arrive at the most accurate HER2 status designation after HER2 FISH equivocal results; however, little is known about its influence on the clinical practice of pathologist. The purpose of this study was to evaluate the impact of the revised 2018 ASCO/CAP guidelines on the HER2 status designation. METHODS: We retrospectively reviewed the HER2 FISH testing results from 2233 cases of invasive breast cancer between January 2014 and December 2017. Concomitant immunohistochemistry (IHC) were performed on the same tissue blocks that were used for the FISH testing. RESULTS: Compared to the 2013 guidelines, the HER2 status in 183 (8.2%) cases were re-defined when reassessed by the 2018 guidelines. Among these 183 cases, 175 equivocal cases according to the 2013 guideline were re-defined as HER2 negative (n = 173) or HER2 positive (n = 2). Eight previously classified as HER2 positive cases were converted to negative in the 2018 scheme, all of which were with HER2 IHC scores of 1+ or 2+. The number of cases in the negative category was 1705 according to the 2018 guidelines as opposed to 1524 by the 2013 guidelines. CONCLUSIONS: The updated 2018 ASCO/CAP guidelines eliminated the FISH equivocal category, which can be attributed to reflex HER2 IHC, and partly ease the dilemma for clinical practice. Reflex IHC for FISH equivocal cases is of prime importance; furthermore, HER2 FISH results were converted from positivity to negativity based on the concomitant IHC results in a small percentage of cases. In all, implementation of the 2018 ASCO/CAP guidelines provides much clearer instructions and recommendations for the HER2 status designation, and thus reduces the risk of misdiagnosis.

TA, GT and AC are significantly under-represented in open reading frames of prokaryotic and eukaryotic protein-coding genes.

Genomes can be considered a combination of 16 dinucleotides. Analysing the relative abundance of different dinucleotides may reveal important features of genome evolution. In present study, we conducted extensive surveys on the relative abundances of dinucleotides in various genomic components of 28 bacterial, 20 archaean, 19 fungal, 24 plant and 29 animal species. We found that TA, GT and AC are significantly under-represented in open reading frames of all organisms and in intergenic regions and introns of most organisms. Specific dinucleotides are of greatly varied usage at different codon positions. The significantly low representations of TA, GT and AC are considered the evolutionary consequences of preventing formation of pre-mature stop codons and of reducing intron-splicing options in candidate primary mRNA sequences. These data suggest that a reduction of TA and GT occurred on both strands of the DNA sequence at an early stage of de novo gene birth. Interestingly, GT and AC are also significantly under-represented in current prokaryotic genomes, suggesting that ancient prokaryotic protein-coding genes might have contained introns. The greatly varied usages of specific dinucleotides at different codon positions are considered evolutionary accommodations to compensate the unavailability of specific codons and to avoid formation of pre-mature stop codons. This is the first report presenting data of dinucleotide relative abundance to indicate the possible existence of spliceosomal introns in ancient prokaryotic genes and to hypothesize early steps of de novo gene birth.