Efficient and multiplexed somatic genome editing with Cas12a mice.

Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations in areas ranging from neuroscience to cancer biology and beyond. However, existing models are limited in their ability to create multiple targeted edits. Thus, our understanding of the complex genetic interactions that underlie development, homeostasis, and disease remains incomplete. Cas12a is an RNA-guided endonuclease with unique attributes that enable simple targeting of multiple genes with crRNA arrays containing tandem guides. To accelerate and expand the generation of complex genotypes in somatic cells, we generated transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a). In these mice, enAsCas12a-mediated somatic genome editing robustly generated compound genotypes, as exemplified by the initiation of diverse cancer types driven by homozygous inactivation of trios of tumor suppressor genes. We further integrated these modular crRNA arrays with clonal barcoding to quantify the size and number of tumors with each array, as well as the efficiency of each crRNA. These Cas12a alleles will enable the rapid generation of disease models and broadly facilitate the high-throughput investigation of coincident genomic alterations in somatic cells in vivo .

Serum binding folate receptor autoantibodies lower in autistic boys and positively-correlated with folate.

Folate receptor autoantibody (FRAA) has caught increasing attention since its discovery in biological fluids of patients with autism spectrum disorder (ASD), but quantification and understanding of its function are still in their infancy. In this study, we aimed to quantify serum binding-FRAA and explore its relation with serum folate, vitamin B12 (VB12) and ferritin. We quantitated serum binding-FRAA in 132 ASD children and 132 typically-developing (TD) children, as well as serum levels of folate, VB12 and ferritin. The results showed that serum binding-FRAA in the ASD group was significantly lower than that in the TD group (p < 0.0001). Further analysis showed that the difference between these two groups was attributed to boys in each group, not girls. There was no statistically significant difference in folate levels between the ASD and TD groups (p > 0.05). However, there was significant difference in boys between these two groups, not girls. Additionally, the combination of nitrite and binding-FRAA showed potential diagnostic value in patients with ASD (AUC > 0.7). Moreover, in the ASD group, the level of folate was consistent with that of binding-FRAA, whereas in the TD group, the binding-FRAA level was high when the folate level was low. Altogether, these differences revealed that the low serum FRAA in autistic children was mediated by multiple factors, which deserves more comprehensive investigation with larger population and mechanistic studies.

Classification of autism spectrum disorder using electroencephalography in Chinese children: a cross-sectional retrospective study.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition characterized by diverse clinical features. EEG biomarkers such as spectral power and functional connectivity have emerged as potential tools for enhancing early diagnosis and understanding of the neural processes underlying ASD. However, existing studies yield conflicting results, necessitating a comprehensive, data-driven analysis. We conducted a retrospective cross-sectional study involving 246 children with ASD and 42 control children. EEG was collected, and diverse EEG features, including spectral power and spectral coherence were extracted. Statistical inference methods, coupled with machine learning models, were employed to identify differences in EEG features between ASD and control groups and develop classification models for diagnostic purposes. Our analysis revealed statistically significant differences in spectral coherence, particularly in gamma and beta frequency bands, indicating elevated long range functional connectivity between frontal and parietal regions in the ASD group. Machine learning models achieved modest classification performance of ROC-AUC at 0.65. While machine learning approaches offer some discriminative power classifying individuals with ASD from controls, they also indicate the need for further refinement.

Associations of unintended pregnancy with autism spectrum disorders and the modification of folic acid supplements.

There is limited evidence on the associations of unintended pregnancy with autism spectrum disorders (ASD). This study aimed to examine this relationship and the modification of pre-conceptional and prenatal folic acid supplements. Six thousand and five toddlers aged 16 to 30 months from seven cities of six provinces in China were eligible for participation. Information on unintended pregnancy and folic acid supplements was obtained via questionnaires from caregivers of toddlers. The diagnosis of ASD was based on the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) and the Chinese version of the Childhood Autism Rating Scale (CARS). Of the 6005 toddlers in the study (3337 boys and 2668 girls), 71 (1.18%) received the diagnosis of ASD. Generalized linear models with a logit link function showed unintended pregnancy was positively associated with ASD (odds ratios [OR] = 1.69, 95% confidence interval [CI], 1.05-2.79). Stratified estimates indicated that the association remained stable among toddlers of mothers without pre-conceptional and prenatal folic acid supplements (OR = 2.75, 95% CI, 1.04-7.27; n = 1243, 20.70%). Unintended pregnancy was associated with higher odds of ASD in 16-30 months of toddlers, and the association was consistent among toddlers of mothers without prenatal folic acid supplements. Our findings emphasize the need to raise awareness of the risk of unintended pregnancy and the benefits of folic acid supplements among Chinese women.

[18F-FDG PET/CT Metabolic Parameters and Circulating Tumour DNA Mutation Abundance in Diffuse Large B-Cell Lymphoma: Correlation and Survival Analysis].

OBJECTIVE: To investigate the correlation between 18Fluoro-deoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) metabolic parameters and peripheral blood circulating tumour DNA (ctDNA) in patients with diffuse large B-cell lymphoma (DLBCL), and the prognostic value of these two types of parameters in predicting progression-free survival (PFS). METHODS: Clinical, PET/CT and ctDNA data of DLBCL patients who underwent peripheral blood ctDNA testing and corresponding PET/CT scans during the same period were retrospectively analyzed. At the time of ctDNA sampling and PET scan, patients were divided into baseline and relapsed/refractory (R/R) groups according to different disease conditions. CtDNA mutation abundance was expressed as variant allele frequency (VAF), including maximum VAF (maxVAF) and mean VAF (meanVAF). Total metabolic tumour volume (TMTV) and total lesion glycolysis (TLG) were obtained by the 41% maximum normalized uptake value method, and the distance between the two farthest lesions (Dmax) was used to assess the correlation between PET parameters and ctDNA mutation abundance using Spearman correlation analysis. The receiver operating characteristic (ROC) curves were used to obtain the optical cut-off values of those parameters in predicting PFS in the baseline and R/R groups, respectively. Survival curves were outlined using the Kaplan-Meier method and log-rank test was performed to compare survival differences. RESULTS: A total of 67 DLBCL patients ï¼»28 males and 39 females, median age 56.0(46.0, 67.0) yearsï¼½ were included and divided into baseline group (29 cases) and R/R group (38 cases). Among these PET parameters, baseline TMTV, TLG, and Dmax were significantly correlated with baseline ctDNA mutation abundance, except for maximum standardized uptake value (SUVmax) (maxVAF vs TMTV: r=0.711; maxVAF vs TLG: r=0.709; maxVAF vs Dmax: r=0.672; meanVAF vs TMTV: r=0.682; meanVAF vs TLG: r=0.677; meanVAF vs Dmax: r=0.646). While in all patients, these correlations became weaker significantly. Among R/R patients, only TMTV had a weak correlation with meanVAF (r=0.376). ROC analysis showed that, the specificity of TMTV, TLG and Dmax in predicting PFS was better than mutation abundance, while the sensitivity of ctDNA mutation abundance was better. Except R/R patients, TMTV, TLG, Dmax, and VAF were significantly different at normal/elevated lactate dehydrogenase in baseline group and all patients (all P<0.05). Survival curves indicated that high TMTV (>109.5 cm3), high TLG (>2 141.3), high Dmax (>33.1 cm) and high VAF (maxVAF>7.74%, meanVAF>4.39%) were risk factors for poor PFS in baseline patients, while only high VAF in R/R patients (both maxVAF and meanVAF >0.61%) was a risk factor for PFS. CONCLUSION: PET-derived parameters correlate well with ctDNA mutation abundance, especially in baseline patients. VAF of ctDNA predicts PFS more sensitively than PET metabolic parameters, while PET metabolic tumour burden with better specificity. TMTV, TLG and VAF all have good prognostic value for PFS. PET/CT combined with ctDNA has potential for further studies in prognostic assessment and personalized treatment.

Chance promoter activities illuminate the origins of eukaryotic intergenic transcriptions.

It is debated whether the pervasive intergenic transcription from eukaryotic genomes has functional significance or simply reflects the promiscuity of RNA polymerases. We approach this question by comparing chance promoter activities with the expression levels of intergenic regions in the model eukaryote Saccharomyces cerevisiae. We build a library of over 105 strains, each carrying a 120-nucleotide, chromosomally integrated, completely random sequence driving the potential transcription of a barcode. Quantifying the RNA concentration of each barcode in two environments reveals that 41-63% of random sequences have significant, albeit usually low, promoter activities. Therefore, even in eukaryotes, where the presence of chromatin is thought to repress transcription, chance transcription is prevalent. We find that only 1-5% of yeast intergenic transcriptions are unattributable to chance promoter activities or neighboring gene expressions, and these transcriptions exhibit higher-than-expected environment-specificity. These findings suggest that only a minute fraction of intergenic transcription is functional in yeast.

A Privacy-Preserving, Two-Party, Secure Computation Mechanism for Consensus-Based Peer-to-Peer Energy Trading in the Smart Grid.

Consumers in electricity markets are becoming more proactive because of the rapid development of demand-response management and distributed energy resources, which boost the transformation of peer-to-peer (P2P) energy-trading mechanisms. However, in the P2P negotiation process, it is a challenging task to prevent private information from being attacked by malicious agents. In this paper, we propose a privacy-preserving, two-party, secure computation mechanism for consensus-based P2P energy trading. First, a novel P2P negotiation mechanism for energy trading is proposed based on the consensus + innovation (C + I) method and the power transfer distribution factor (PTDF), and this mechanism can simultaneously maximize social welfare and maintain physical network constraints. In addition, the C + I method only requires a minimum set of information to be exchanged. Then, we analyze the strategy of malicious neighboring agents colluding to attack in order to steal private information. To defend against this attack, we propose a two-party, secure computation mechanism in order to realize safe negotiation between each pair of prosumers based on Paillier homomorphic encryption (HE), a smart contract (SC), and zero-knowledge proof (ZKP). The energy price is updated in a safe way without leaking any private information. Finally, we simulate the functionality of the privacy-preserving mechanism in terms of convergence performance, computational efficiency, scalability, and SC operations.

Metabolomics and proteomics revealed the synthesis difference of aroma precursors in tobacco leaves at various growth stages.

Tobacco has a high economic value as the largest cash crop worldwide. The quality of flue-cured tobacco is closely related to the overall status of compounds in fresh tobacco leaves, and the aroma precursor plays a key role in the aroma quality of flue-cured tobacco. The untargeted metabolomics and label-free quantitative proteomics analysis of tobacco leaves in three growth stages (root stretching, prosperous growth, and maturation) retrieved 243 metabolites and 4313 proteins (944 differentially expressed proteins), which showed that carbohydrate, amino acid, and fatty acid metabolism varies among the three growth stages. Also, the most of amino acids, organic acids, fatty acids, and polyphenols reduced in the vegetative growth stage, while increased in the reproductive growth stage. On the other hand, alkaloids such as nicotine, nornicotine, and anatabine increased continuously in tobacco leaves during the three growth stages. This study helps us understand the growth and development characteristics of Yun87 flue-cured tobacco in the field before harvest, and it provides a certain omics basis for the industrial crop flue-cured tobacco.

Proteomic and Metabolomic Revealed Differences in the Distribution and Synthesis Mechanism of Aroma Precursors in Yunyan 87 Tobacco Leaf, Stem, and Root at the Seedling Stage.

Tobacco, as an important cash crop and model plant, has been the subject of various types of research. The quality of flue-cured tobacco products depends on the compound collection of tobacco leaves, including pigments, carbohydrates, amino acids, polyphenols, and alkaloid aroma precursors. The present study investigates tobacco seedling organs (leaf, stem, and root) with the assistance of label-free proteomic technology and untargeted metabonomic technology. We analyzed 4992 proteins and 298 metabolites obtained in the leaf, stem, and root groups and found that there were significant differences in both primary and secondary metabolism processes involved in aroma precursor biosynthesis, such as carbohydrate metabolism, energy metabolism, and amino acid biosynthesis, and phenylpropanoid, flavonoid, and alkaloid biosynthesis. The findings showed that the contents of alkaloid metabolites such as nornicotine, anatabine, anatalline, and myosmine were significantly higher in tobacco roots than in leaves and stems at the seedling stage.

Exploring brainstem auditory evoked potentials and mental development index as early indicators of autism spectrum disorders in high-risk infants.

This study of infants from Hubei Province, China examined brainstem auditory evoked potentials (BAEP) and mental development index (MDI) as possible early indicators associated with autism spectrum disorders (ASD). The 34 ASD cases and 102 controls who had recovered from perinatal conditions were matched for age, sex, gestational age, birth weight and maternal age. BAEP absolute latencies (AL) I, III, V and interpeak latencies (IPL) I-III, III-V, I-V were compared in ASD cases and controls at ages 1, 3 and 6 months. MDI scores were compared in these infants from 1 month to 2 years old. Multiple logistic regression analysis was performed to test associations among ASD, BAEP and MDI. Results showed BAEP AL I, V and IPL III-V prolonged in the ASD group (p < 0.001), and MDI scores in ASD cases sharply declining from 12 to 24 months (p < 0.001). Regression analysis revealed odds ratios (OR) indicating that ASD was likely associated with abnormal values of BAEP AL I at 1 and 3 months (ORAL I : 4.27; ORAL I : 4.13), and AL V at 6 months (ORAL V : 7.85). Lower MDI scores (MDI < 80) in infants at 1, 3, and 6 months were likely associated with ASD (ORMDI : 2.58; ORMDI : 3.83; ORMDI : 4.87). These data show that abnormal BAEP values and low MDI scores are independent factors associated with ASD, and that monitoring of BAEP and MDI during infancy might facilitate screening for ASD development.

Rice MPK17 Plays a Negative Role in the Xa21-Mediated Resistance Against Xanthomonas oryzae pv. oryzae.

Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases affecting rice production worldwide. Xa21 was the first disease resistance gene cloned in rice, which encodes a receptor kinase and confers broad resistance against Xoo stains. Dozens of components in the Xa21-mediated pathway have been identified in the past decades, however, the involvement of mitogen-activated protein kinase (MAPK) genes in the pathway has not been well described. To identify MAPK involved in Xa21-mediated resistance, the level of MAPK proteins was profiled using Western blot analysis. The abundance of OsMPK17 (MPK17) was found decreased during the rice-Xoo interaction in the background of Xa21. To investigate the function of MPK17, MPK17-RNAi and over-expression (OX) transgenic lines were generated. The RNAi lines showed an enhanced resistance, while OX lines had impaired resistance against Xoo, indicating that MPK17 plays negative role in Xa21-mediated resistance. Furthermore, the abundance of transcription factor WRKY62 and pathogenesis-related proteins PR1A were changed in the MPK17 transgenic lines when inoculated with Xoo. We also observed that the MPK17-RNAi and -OX rice plants showed altered agronomic traits, indicating that MPK17 also plays roles in the growth and development. On the basis of the current study and published results, we propose a "Xa21-MPK17-WRKY62-PR1A" signaling that functions in the Xa21-mediated disease resistance pathway. The identification of MPK17 advances our understanding of the mechanism underlying Xa21-mediated immunity, specifically in the mid- and late-stages.

Comparison on anaerobic phosphorus release and recovery from waste activated sludge by different chemical pretreatment methods: Focus on struvite quality and benefit analysis.

Phosphorus recovery from waste activated sludge (WAS) is expected to alleviate the shortage of phosphate rock and reduce eutrophication. In this study, acid, alkali and sodium polyacrylate (PAAS) were compared to enhance phosphorus release and recovery from WAS. During anaerobic fermentation (AF) stage, the optimal pretreated conditions for ortho-phosphate release were the pH of 4 (AF 12 h), 13 (AF 12 h) and 22.4 g PAAS/L (AF 24 h) with the phosphorus release efficiencies of 40.9%, 62.6% and 31.7%, respectively. Acid, alkali and PAAS addition were beneficial for apatite phosphorus (AP), non-apatite inorganic phosphorus (NAIP) and organic phosphorus (OP) release from WAS, respectively. Strong acidic (pH = 4) and alkaline (pH = 12 and 13) conditions inhibited the release of soluble ammonia, while PAAS would not have a negative impact on the release of soluble ammonia. By means of precipitation crystallization, the ortho-phosphate could be almost recovered after acid/alkali pretreatment compared with PAAS (88.9%) at optimal Mg/P molar ratio of 1.5:1. The XRD, FT-IR and SEM-EDX analyses confirmed the main component in the product was struvite. The purity of the struvite in the product recovered from acid (named PreAC, 78.9%) and alkali (named PreAL, 89.6%) pretreated sludge were higher than that of the PAAS (named PrePA, 72.3%) by elemental analysis. The mercury and chromium content existed in PreAC were above the Control Standards of Pollutants in Sludge for Agricultural Use, whereas detected heavy metal elements level of the PreAL and PrePA were below the standard. By means of cost analysis, acid/alkali pretreatment could obtain economic benefits compared with PAAS. Thus, those discoveries would broaden the phosphorus recovery way to serve in practice.

18F-FDG PET/CT Findings in a Woman With Kaposiform Hemangioendothelioma in the Sacrum.

ABSTRACT: Kaposiform hemangioendothelioma is a rare vascular tumor with borderline malignancy and is typically diagnosed in infancy or early childhood. It most commonly affects cutaneous tissues, whereas the subtype with only primary bone involvement is extremely rare. Herein, we report a case of Kaposiform hemangioendothelioma involving the sacrum in a 37-year-old woman, with intense 18F-FDG accumulation in the lytic lesion on PET/CT. This case indicates that Kaposiform hemangioendothelioma with the primary bone involvement should be taken into consideration as a rare differential diagnosis for lytic lesions with increased 18F-FDG uptake on PET/CT.

Complete chloroplast genome sequence of Mucuna prurien and its phylogenetic position.

Mucuna pruriens is traditional medicinal plant originated in South Africa. We characterize the complete plastid genome of M. pruriens, which is a circular-mapping molecule 152,119 bp in length. The genome has a large single-copy region (LSC) of 78,258 bp and a small single-copy region (SSC) of 18,735 bp, respectively. Additionally, the overall GC content of the chloroplast genome was 35.37%. The genome contains 138 genes, including 96 protein-coding, 38 tRNA, and four rRNA genes. The gene content and structure are conserved compared to other species in the genus Glycine. The chloroplast genome and existing data were used to infer its phylogenetic position. The results showed that M. pruriens clustered together with Glycine max and G. soja. These findings provide potential genetic markers that can aid in understanding the genetic diversity of M. pruriens.

Is the Genetic Code Optimized for Resource Conservation?

The causes and consequences of the nonrandom structure of the standard genetic code (SGC) have been of long-standing interest. A recent study reported that mutations in present-day protein-coding sequences are less likely to increase proteomic nitrogen and carbon uses under the SGC than under random genetic codes, concluding that the SGC has been selectively optimized for resource conservation. If true, this finding might offer important information on the environment in which the SGC and some of the earliest life forms evolved. However, we here show that the hypothesis of optimization of a genetic code for resource conservation is theoretically untenable. We discover that the aforementioned study estimated the expected mutational effect by inappropriately excluding mutations lowering resource consumptions and including mutations involving stop codons. After remedying these problems, we find no evidence that the SGC is optimized for nitrogen or carbon conservation.

Differences in SCN1A intronic variants result in diverse aberrant splicing patterns and are related to the phenotypes of epilepsy with febrile seizures.

OBJECTIVE: Intronic variants of the SCN1A gene are detected in patients with epilepsy with febrile seizures (EFS), which includes a series of phenotypes with different severities. However, the pathogenicity of intronic variants and their genotype-phenotype correlation remain under characterized. The purpose of this study was to determine the changes in mRNA splicing caused by SCN1A intronic variants associated with EFS and their association with phenotypes. METHODS: Five SCN1A intronic variants detected in patients with focal epilepsy with antecedent febrile seizures plus (FEFS+) and Dravet syndrome (DS) were molecularly cloned. Through an in vitro minigene splicing assay, their influence on mRNA splicing was qualitatively and quantitatively compared and analyzed using reverse-transcription polymerase chain reaction (RT-PCR) and fluorescence quantitative PCR (Q-PCR). RESULTS: The severe phenotype of DS-associated variants c.602 + 1G > A and c.4853-1G > C, which occurred in canonical splice sites of introns, caused exon skipping and little retention of full-length mRNA, while the milder phenotype of FEFS+-associated variants c.473 + 5G > A, c.473 + 5G > C and c.4853-25T > A, which occurred in potential splice sites or in deep intronic regions, presented partial exon skipping or intronic insertion and significantly higher retention of full-length mRNA at different levels. Full-length mRNA retention was negatively correlated with the location of intronic variants and phenotype severity. CONCLUSION: The different aberrant splicing patterns resulting from SCN1A intronic variants with different positions represent a potential molecular mechanism for phenotypic differences in EFS. This research provides valuable clues for functional studies on the pathogenicity of intronic variants and for the evaluation of genotype-phenotype correlations.

[Combination of apparent diffusion coefficient and amplitude-integrated electroencephalogram to predict the outcome of comatose patients with severe traumatic brain injury].

OBJECTIVE: To examine whether the combination of quantitative regional apparent diffusion coefficient (ADC) and amplitude-integrated electroencephalogram (aEEG) can predict the outcome of comatose patients with severe traumatic brain injury (sTBI). METHODS: A prospective study was conducted. The patients with coma caused by sTBI [Glasgow coma scale (GCS) < 8] admitted to Suqian First Hospital from January 2016 to June 2019 were enrolled. All patients underwent aEEG examination and magnetic resonance imaging (MRI) scan within 1 week after emergency treatment. The ADC values of 9 regions of interest (frontal gray matter and white matter, parietal gray matter and white matter, temporal gray matter and white matter, caudate nucleus of basal ganglia, lenticular nucleus and thalamus) were measured by head MRI, and the mean ADC values of frontal lobe, parietal lobe, temporal lobe and basal ganglia were calculated respectively. According to the follow-up results after 12 months, the differences of each index between patients with poor prognosis [Glasgow outcome score (GOS) 1-2] and patients with good prognosis (GOS 3-5) were compared; the receiver operating characteristic curve (ROC curve) was drawn to evaluate the predictive ability of aEEG and ADC for the good prognosis of patients with sTBI, and the predictive value of the combination of aEEG and ADC. RESULTS: A total of 52 patients with sTBI were enrolled, with mean age of (36.7±13.9) years old, 35 of whom were male. Within 12 months follow-up, 29 patients had achieved favorable outcomes and 23 patients had unfavorable outcome. There were 21, 17 and 14 patients with aEEG, and grade, respectively, and 19, 10 and 0 patients had good prognosis respectively. ADC values of 9 regions of interest in patients with good prognosis were significantly higher than those in patients with poor prognosis (×10-6 mm2/s: 924±107 vs. 531±87 in frontal gray matter, 804±95 vs. 481±74 in frontal white matter, 831±93 vs. 683±72 in temporal gray matter, 726±87 vs. 654±63 in temporal white matter, 767±79 vs. 690±75 in parietal gray matter, 716±84 vs. 642±62 in parietal white matter, 689±70 vs. 465±68 in caudate nucleus, 723±84 vs. 587±71 in lenticular nucleus, 807±79 vs. 497±67 in thalamus, all P < 0.01). ROC curve analysis showed that the area under ROC curve (AUC) of aEEG for predicting good prognosis of sTBI patients was 0.826, when the cut-off value of aEEG was < 1.5, the sensitivity was 94.7% and the specificity was 72.8%. Among the ADC value prediction abilities in the interested areas, the prediction of ADC value in frontal lobe and basal ganglia area were better than that in sTBI patients. AUC was 0.817 and 0.903 respectively. The best cut-off values were > 726×10-6 mm2/s and > 624×10-6 mm2/s respectively, the sensitivity of predicting prognosis were both 100%, and the specificity was 63.4% and 61.8%. A model combining frontal ADC and basal ganglia ADC with aEEG was 91.0% sensitive and 93.7% specific for favorable outcome of sTBI patients. CONCLUSIONS: Combination of the quantitative measurement of regional ADC and aEEG may be useful for predicting the outcome of the patients with sTBI.

On the Origin of Frameshift-Robustness of the Standard Genetic Code.

The standard genetic code (SGC) has been extensively analyzed for the biological ramifications of its nonrandom structure. For instance, mismatch errors due to point mutation or mistranslation have an overall smaller effect on the amino acid polar requirement under the SGC than under random genetic codes (RGCs). A similar observation was recently made for frameshift errors, prompting the assertion that the SGC has been shaped by natural selection for frameshift-robustness-conservation of certain amino acid properties upon a frameshift mutation or translational frameshift. However, frameshift-robustness confers no benefit because frameshifts usually create premature stop codons that cause nonsense-mediated mRNA decay or production of nonfunctional truncated proteins. We here propose that the frameshift-robustness of the SGC is a byproduct of its mismatch-robustness. Of 564 amino acid properties considered, the SGC exhibits mismatch-robustness in 93-133 properties and frameshift-robustness in 55 properties, respectively, and that the latter is largely a subset of the former. For each of the 564 real and 564 randomly constructed fake properties of amino acids, there is a positive correlation between mismatch-robustness and frameshift-robustness across one million RGCs; this correlation arises because most amino acid changes resulting from a frameshift are also achievable by a mismatch error. Importantly, the SGC does not show significantly higher frameshift-robustness in any of the 55 properties than RGCs of comparable mismatch-robustness. These findings support that the frameshift-robustness of the SGC need not originate through direct selection and can instead be a site effect of its mismatch-robustness.

A SWEET surprise: Anaerobic fungal sugar transporters and chimeras enhance sugar uptake in yeast.

In the yeast Saccharomyces cerevisiae, microbial fuels and chemicals production on lignocellulosic hydrolysates is constrained by poor sugar transport. For biotechnological applications, it is desirable to source transporters with novel or enhanced function from nonconventional organisms in complement to engineering known transporters. Here, we identified and functionally screened genes from three strains of early-branching anaerobic fungi (Neocallimastigomycota) that encode sugar transporters from the recently discovered Sugars Will Eventually be Exported Transporter (SWEET) superfamily in Saccharomyces cerevisiae. A novel fungal SWEET, NcSWEET1, was identified that localized to the plasma membrane and complemented growth in a hexose transporter-deficient yeast strain. Single cross-over chimeras were constructed from a leading NcSWEET1 expression-enabling domain paired with all other candidate SWEETs to broadly scan the sequence and functional space for enhanced variants. This led to the identification of a chimera, NcSW1/PfSW2:TM5-7, that enhanced the growth rate significantly on glucose, fructose, and mannose. Additional chimeras with varied cross-over junctions identified residues in TM1 that affect substrate selectivity. Furthermore, we demonstrate that NcSWEET1 and the enhanced NcSW1/PfSW2:TM5-7 variant facilitated novel co-consumption of glucose and xylose in S. cerevisiae. NcSWEET1 utilized 40.1% of both sugars, exceeding the 17.3% utilization demonstrated by the control HXT7(F79S) strain. Our results suggest that SWEETs from anaerobic fungi are beneficial tools for enhancing glucose and xylose co-utilization and offers a promising step towards biotechnological application of SWEETs in S. cerevisiae.