Toxic antiphage defense proteins inhibited by intragenic antitoxin proteins.

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here, we report that these proteins are toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic RpnL. The crystal structure of RpnAS revealed a dimerization interface encompassing α helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document that plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose that many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms.

The three-sided right-handed ß-helix is a versatile fold for glycan interactions.

Interactions between proteins and glycans are critical to various biological processes. With databases of carbohydrate-interacting proteins and increasing amounts of structural data, the three-sided right-handed ß-helix (RHBH) has emerged as a significant structural fold for glycan interactions. In this review, we provide an overview of the sequence, mechanistic, and structural features that enable the RHBH to interact with glycans. The RHBH is a prevalent fold that exists in eukaryotes, prokaryotes, and viruses associated with adhesin and carbohydrate-active enzyme (CAZyme) functions. An evolutionary trajectory analysis on structurally characterized RHBH-containing proteins shows that they likely evolved from carbohydrate-binding proteins with their carbohydrate-degrading activities evolving later. By examining three polysaccharide lyase and three glycoside hydrolase structures, we provide a detailed view of the modes of glycan binding in RHBH proteins. The 3-dimensional shape of the RHBH creates an electrostatically and spatially favorable glycan binding surface that allows for extensive hydrogen bonding interactions, leading to favorable and stable glycan binding. The RHBH is observed to be an adaptable domain capable of being modified with loop insertions and charge inversions to accommodate heterogeneous and flexible glycans and diverse reaction mechanisms. Understanding this prevalent protein fold can advance our knowledge of glycan binding in biological systems and help guide the efficient design and utilization of RHBH-containing proteins in glycobiology research.

Evolink: a phylogenetic approach for rapid identification of genotype-phenotype associations in large-scale microbial multispecies data.

MOTIVATION: The discovery of the genetic features that underly a phenotype is a fundamental task in microbial genomics. With the growing number of microbial genomes that are paired with phenotypic data, new challenges, and opportunities are arising for genotype-phenotype inference. Phylogenetic approaches are frequently used to adjust for the population structure of microbes but scaling them to trees with thousands of leaves representing heterogeneous populations is highly challenging. This greatly hinders the identification of prevalent genetic features that contribute to phenotypes that are observed in a wide diversity of species. RESULTS: In this study, Evolink was developed as an approach to rapidly identify genotypes associated with phenotypes in large-scale multispecies microbial datasets. Compared with other similar tools, Evolink was consistently among the top-performing methods in terms of precision and sensitivity when applied to simulated and real-world flagella datasets. In addition, Evolink significantly outperformed all other approaches in terms of computation time. Application of Evolink on flagella and gram-staining datasets revealed findings that are consistent with known markers and supported by the literature. In conclusion, Evolink can rapidly detect phenotype-associated genotypes across multiple species, demonstrating its potential to be broadly utilized to identify gene families associated with traits of interest. AVAILABILITY AND IMPLEMENTATION: The source code, docker container, and web server for Evolink are freely available at https://github.com/nlm-irp-jianglab/Evolink.

Nonlinear metasurface engineering with disordered gold nanorods on ITO: a cost-effective approach to broadband response, polarization-independence, and weak nonlinear index dispersion.

The strong coupling of epsilon-near-zero materials with nanoantennas has demonstrated enhanced nonlinear optical responses, yet practical challenges persist. Here, we propose an alternative: an ultrathin metasurface featuring broadband response with a weakly dispersive nonlinear index, achieved through a simple implementation. Our metasurface, comprising a disordered gold nanorod array on indium tin oxide, exhibits polarization-independent behavior and a large average nonlinear refractive index of 5 cm2/GW across a broad wavelength range (1000-1300 nm). Enhanced performance is attributed to the weak coupling between gold nanorods and indium tin oxide, offering a cost-effective method for nonlinear optical metasurfaces and a flexible design in nanophotonic applications.

Differential diagnosis of breast mucinous carcinoma with an oval shape from fibroadenoma based on ultrasonographic features.

BACKGROUND: Approximately 50% of breast mucinous carcinomas (MCs) are oval and have the possibility of being misdiagnosed as fibroadenomas (FAs). We aimed to identify the key features that can help differentiate breast MC with an oval shape from FA on ultrasonography (US). METHODS: Seventy-six MCs from 71 consecutive patients and 50 FAs with an oval shape from 50 consecutive patients were included in our study. All lesions pathologically diagnosed. According to the Breast Imaging Reporting and Data System (BI-RADS), first, the ultrasonographic features of the MCs and FAs were recorded and a final category was assessed. Then, the differences in ultrasonographic characteristics between category 4 A (low-risk group) and category 4B-5 (medium-high- risk group) MCs were identified. Finally, other ultrasonographic features of MC and FA both with an oval shape were compared to determine the key factors for differential diagnosis. The Mann-Whitney test, χ2 test or Fisher's exact test was used to compare data between groups. RESULTS: MCs with an oval shape (81.2%) and a circumscribed margin (25%) on US were more commonly assessed in the low-risk group (BI-RADS 4 A) than in the medium-high-risk group (BI-RADS 4B-5) (20%, p < 0.001 and 0%, p = 0.001, respectively). Compared with those with FA, patients with MC were older, and tended to have masses with non-hypoechoic patterns, not circumscribed margins, and a posterior echo enhancement on US (p < 0.001, p < 0.001, and p = 0.003, respectively). CONCLUSION: The oval shape was the main reason for the underestimation of MCs. On US, an oval mass found in the breast of women of older age with non-hypoechoic patterns, not circumscribed margins, and a posterior echo enhancement was associated with an increased risk of being an MC, and should be subjected to active biopsy.

Protein Kinase N1 Level Predicts Acute Kidney Injury in Patients Undergoing Cardiac Surgery: A Prospective Cohort Study.

INTRODUCTION: The objective of this study was to examine the utility of protein kinase N1 (PKN1) as a biomarker of cardiac surgery-associated AKI (CSA-AKI). METHODS: A prospective cohort study of 110 adults undergoing on-pump cardiac surgery was conducted. The associations between post-operative PKN1 and CSA-AKI, AKI severity, need for renal replacement therapy (RRT), duration of AKI, length of ICU stay, and post-operative hospital stay were evaluated. RESULTS: Patients were categorized into three groups according to PKN1 tertiles. The incidence of CSA-AKI in the third tertile was 3.4-fold higher than that in the first. PKN1 was an independent risk factor for CSA-AKI. The discrimination of PKN1 to CSA-AKI assessed by ROC curve indicated that the AUC was 0.70, and the best cutoff was 5.025 ng/mL. This group (>5.025 ng/mL) was more likely to develop CSA-AKI (p < 0.001). The combined AUC of EuroSCORE, aortic cross-clamp time, and PKN1 was 0.82 (p < 0.001). A higher level of PKN1 was related to increased need for RRT, longer duration of AKI, and length of ICU and post-operative hospital stays. CONCLUSIONS: PKN1 could be a potential biomarker for the prediction of CSA-AKI. The combination of PKN1, EuroSCORE, and aortic cross-clamp time was likely to predict the occurrence of CSA-AKI.

KH domain proteins: Another family of bacterial RNA matchmakers?

In many bacteria, the stabilities and functions of small regulatory RNAs (sRNAs) that act by base pairing with target RNAs most often are dependent on Hfq or ProQ/FinO-domain proteins, two classes of RNA chaperone proteins. However, while all bacteria appear to have sRNAs, many have neither Hfq nor ProQ/FinO-domain proteins raising the question of whether another factor might act as an sRNA chaperone in these organisms. Several recent studies have reported that KH domain proteins, such as KhpA and KhpB, bind sRNAs. Here we describe what is known about the distribution, structures, RNA-binding properties, and physiologic roles of KhpA and KhpB and discuss evidence for and against these proteins serving as sRNAs chaperones.

Gut Microbiome-Wide Search for Bacterial Azoreductases Reveals Potentially Uncharacterized Azoreductases Encoded in the Human Gut Microbiome.

The human gut is home to trillions of microorganisms that are responsible for the modification of many orally administered drugs, leading to a wide range of therapeutic outcomes. Prodrugs bearing an azo bond are designed to treat inflammatory bowel disease and colorectal cancer via microbial azo reduction, allowing for topical application of therapeutic moieties to the diseased tissue in the intestines. Despite the inextricable link between microbial azo reduction and the efficacy of azo prodrugs, the prevalence, abundance, and distribution of azoreductases have not been systematically examined across the gut microbiome. Here, we curated and clustered amino acid sequences of experimentally confirmed bacterial azoreductases and conducted a hidden Markov model-driven homolog search for these enzymes across 4644 genome sequences present in the representative Unified Human Gastrointestinal Genomes collection. We identified 1958 putative azo-reducing species, corroborating previous findings that azo reduction appears to be a ubiquitous function of the gut microbiome. However, through a systematic comparison of predicted and confirmed azo-reducing strains, we hypothesize the presence of uncharacterized azoreductases in 25 prominent strains of the human gut microbiome. Finally, we confirmed the azo reduction of Acid Orange 7 by multiple strains of Fusobacterium nucleatum, Bacteroides fragilis, and Clostridium clostridioforme Together, these results suggest the presence and activity of many uncharacterized azoreductases in the human gut microbiome and motivate future studies aimed at characterizing azoreductase genes in prominent members of the human gut microbiome. SIGNIFICANCE STATEMENT: This work systematically examined the prevalence, abundance, and distribution of azoreductases across the healthy and inflammatory bowel disease human gut microbiome, revealing potentially uncharacterized azoreductase genes. It also confirmed the reduction of Acid Orange 7 by strains of Fusobacterium nucleatum, Bacteroides fragilis, and Clostridium clostridioforme.

Boosting optical nonlinearity in epsilon-near-zero trilayer coatings.

Materials with large optical nonlinearity are highly desired for various applications such as all-optical signal processing and storage. Recently, indium tin oxide (ITO) has been found to possess strong optical nonlinearity in the spectral region where its permittivity vanishes. Here, we demonstrate that ITO/Ag/ITO trilayer coatings, deposited by magnetron sputtering with high-temperature heat treatment, can significantly enhance the nonlinear response in their effective epsilon-near-zero (ENZ) regions. The obtained results show that the carrier concentrations of our trilayer samples can reach 7.25 × 1021 cm-3, and the ENZ region can shift to the spectrum close to the visible range. In the ENZ spectral region, the ITO/Ag/ITO samples exhibit enhanced nonlinear refractive indices as large as 2.397 × 10-15 m2 W-1, over 27 times larger than that of an individual ITO layer. Such a nonlinear optical response is well described using a two-temperature model. Our findings provide a new paradigm for developing nonlinear optical devices for applications requiring low power.

Taxonomic distribution and evolutionary analysis of the equol biosynthesis gene cluster.

BACKGROUND: Equol, an isoflavonoid metabolite with possible health benefits in humans, is known to be produced by some human gut bacteria. While the genes encoding the equol production pathway have been characterized in a few bacterial strains, a systematic analysis of the equol production pathway is currently lacking. RESULTS: This study presents an analysis of the taxonomic distribution and evolutionary history of the gene cluster encoding the equol production pathway. A survey for equol gene clusters within the Genome Taxonomy Database bacterial genomes and human gut metagenomes resulted in the identification of a highly conserved gene cluster found in nine bacterial species from the Eggerthellaceae family. The identified gene clusters from human gut metagenomes revealed potential variations in the equol gene cluster organization and gene content within the equol-producing Eggerthellaceae clades. Subsequent analysis showed that in addition to the four genes directly involved in equol production, multiple other genes were consistently found in the equol gene clusters. These genes were predicted to encode a putative electron transport complex and hydrogenase maturase system, suggesting potential roles for them in the equol production pathway. Analysis of the gene clusters and a phylogenetic reconstruction of a putative NAD kinase gene provided evidence of the recent transfer of the equol gene cluster from a basal Eggerthellaceae species to Slackia_A equolifaciens, Enteroscipio sp000270285, and Lactococcus garvieae 20-92. CONCLUSIONS: This analysis demonstrates that the highly conserved equol gene cluster is taxonomically restricted to the Eggerthellaceae family of bacteria and provides evidence of the role of horizontal gene transfer in the evolutionary history of these genes. These results provide a foundation for future studies of equol production in the human gut and future efforts related to bioengineering and the use of equol-producing bacteria as probiotics.

Characterizing Transcriptional Regulatory Sequences in Coronaviruses and Their Role in Recombination.

Novel coronaviruses, including SARS-CoV-2, SARS, and MERS, often originate from recombination events. The mechanism of recombination in RNA viruses is template switching. Coronavirus transcription also involves template switching at specific regions, called transcriptional regulatory sequences (TRS). It is hypothesized but not yet verified that TRS sites are prone to recombination events. Here, we developed a tool called SuPER to systematically identify TRS in coronavirus genomes and then investigated whether recombination is more common at TRS. We ran SuPER on 506 coronavirus genomes and identified 465 TRS-L and 3,509 TRS-B. We found that the TRS-L core sequence (CS) and the secondary structure of the leader sequence are generally conserved within coronavirus genera but different between genera. By examining the location of recombination breakpoints with respect to TRS-B CS, we observed that recombination hotspots are more frequently colocated with TRS-B sites than expected.

Predicting the risk of axillary lymph node metastasis in early breast cancer patients based on ultrasonographic-clinicopathologic features and the use of nomograms: a prospective single-center observational study.

OBJECTIVES: The purpose of this study was to establish two preoperative nomograms to evaluate the risk for axillary lymph node (ALN) metastasis in early breast cancer patients based on ultrasonographic-clinicopathologic features. METHODS: We prospectively evaluated 593 consecutive female participants who were diagnosed with cT1-3N0-1M0 breast cancer between March 2018 and May 2019 at Sun Yat-Sen Memorial Hospital. The participants were randomly classified into training and validation sets in a 4:1 ratio for the development and validation of the nomograms, respectively. Multivariate logistic regression analysis was performed to identify independent predictors of ALN status. We developed Nomogram A and Nomogram B to predict ALN metastasis (presence vs. absence) and the number of metastatic ALNs (≤ 2 vs. > 2), respectively. RESULTS: A total of 528 participants were evaluated in the final analyses. Multivariable analysis revealed that the number of suspicious lymph nodes, long axis, short-to-long axis ratio, cortical thickness, tumor location, and histological grade were independent predictors of ALN status. The AUCs of nomogram A in the training and validation groups were 0.83 and 0.78, respectively. The AUCs of nomogram B in the training and validation groups were 0.87 and 0.87, respectively. Both nomograms were well-calibrated. CONCLUSION: We developed two preoperative nomograms that can be used to predict ALN metastasis (presence vs. absence) and the number of metastatic ALNs (≤ 2 vs. > 2) in early breast cancer patients. Both nomograms are useful tools that will help clinicians predict the risk of ALN metastasis and facilitate therapy decision-making about axillary surgery. KEY POINTS: • We developed two preoperative nomograms to predict axillary lymph node status based on ultrasonographic-clinicopathologic features. • Nomogram A was used to predict axillary lymph node metastasis (presence vs. absence). The AUCs in the training and validation groups were 0.83 and 0.78, respectively. Nomogram B was used to estimate the number of metastatic lymph nodes ( ≤ 2 vs. > 2). The AUCs in the training and validation group were 0.87 and 0.87, respectively. • Our nomograms may help clinicians weigh the risks and benefits of axillary surgery more appropriately.

The taxonomic distribution of histamine-secreting bacteria in the human gut microbiome.

BACKGROUND: Biogenic histamine plays an important role in immune response, neurotransmission, and allergic response. Although endogenous histamine production has been extensively studied, the contributions of histamine produced by the human gut microbiota have not been explored due to the absence of a systematic annotation of histamine-secreting bacteria. RESULTS: To identify the histamine-secreting bacteria from in the human gut microbiome, we conducted a systematic search for putative histamine-secreting bacteria in 36,554 genomes from the Genome Taxonomy Database and Unified Human Gastrointestinal Genome catalog. Using bioinformatic approaches, we identified 117 putative histamine-secreting bacteria species. A new three-component decarboxylation system including two colocalized decarboxylases and one transporter was observed in histamine-secreting bacteria among three different phyla. We found significant enrichment of histamine-secreting bacteria in patients with inflammatory bowel disease but not in patients with colorectal cancer suggesting a possible association between histamine-secreting bacteria and inflammatory bowel disease. CONCLUSIONS: The findings of this study expand our knowledge of the taxonomic distribution of putative histamine-secreting bacteria in the human gut.

PVA-SM microstructure enhanced ratiometric fluorescence probe for formaldehyde detection in solution and gas.

Formaldehyde (FA) is one of the most common pollutants, which has tremendous harm to humans and environment. In this work, 4-amino-3-pentene-2-one (Fluoral-p) and SiO2 coated quantum dot (QD@SiO2) were combined to implement a new ratiometric fluorescence probe QD@SiO2-Fluoral-p for FA detection. In addition, by utilization of polyvinyl alcohol (PVA) and SiO2 microsphere (SM), a kind of PVA-SM microstructure was assembled with QD@SiO2-Fluoral-p to composite a signal enhanced sensing film. The QD@SiO2-Fluoral-p exhibited good response to 0-400 mg/L FA solution and an enhancement around 15 folds was realized after introducing PVA-SM. In both situations, the probe showed linear relationship to FA concentration (CFA), with detection limits of 14 and 0.5 mg/L, respectively. Also, the sensing film showed a good linear response to FA gas in the range of 0 to 2 ppm, with a detection limit 0.03 ppm. As a result, the PVA-SM enhanced ratiometric fluorescence probe features high sensitivity, low detection limit, good selectivity, as well as portable, which can serve as a useful tool for investigating FA in solution and gas at room temperature.

Photothermal microfluidic-assisted self-cleaning effect for a highly reusable SERS sensor.

The synergistic integration of optofluidic and surface enhanced Raman scattering (SERS) sensing is a new analytical technique that provides a number of unique characteristics for enhancing the sensing performance and simplifying the design of microsystems. Here, we propose a reusable optofluidic SERS sensor by integrating Au nanoisland substrate (AuNIS)-coated fiber into a microfluidic chip. Through both systematic experimental and theoretical analysis, the sensor enables efficient self-cleaning based on its optical-to-heat-hydrodynamic energy conversion property. Besides, the sensor exhibits the instrument detection limit down to 10-13mol/L and enhancement factor of 106 for Rhodamine 6G. Our optofluidic SERS sensor with such a photothermal microfluidic-assisted self-cleaning method has the advantages of portability, simple operation, and high cleaning efficiency, which will provide a new, to the best of our knowledge, concept and approach for cost-effective and reusable sensors.

Vortices-interaction-induced microstreaming for the pump-free separation of particles.

Microfluidic techniques have emerged as promising strategies for a wide variety of synthetic or biological sorting. Unfortunately, there is still a lack of sorting with automatic and handy operation. In contrast to passively generated vortices, the thermocapillary vortices produced by temperature gradient have the advantages of flexible manipulation, stable strength, and simple integration. In this Letter, we present a device used for the pump-free separation of particles through vortices interaction without external fluidic control systems required for the majority of existing devices. Specifically, the device induces a different flow type upon the actuation of optical power, and the flow functions, such as simultaneous pumping and sorting, agree with stimulation results very well. More importantly, our developed sorting device can achieve separations by means of tunable cutoff diameter size. Therefore, this versatile device can be utilized to sort complex samples with the advantages of portability, user-friendly control, and automation.

Ultrasound-Guided Vacuum-assisted Biopsy Versus Surgical Resection in Patients With Breast Desmoid Tumor.

BACKGROUND: Recent studies suggest that desmoid tumors can be managed more conservatively rather than undergoing wide surgical resection (SR). Ultrasound-guided vacuum-assisted biopsy (UGVAB) is a minimally invasive technique. This retrospective study aimed to compare the outcome in patients with breast desmoid tumor (BDT) who received UGVAB alone versus SR. MATERIALS AND METHODS: The pathology database was searched for patients diagnosed with BDT ≤ 3 cm from 2007 to 2019. All patients underwent breast ultrasound examination and were then performed UGVAB alone or local SR. The Kaplan-Meier method with a log-rank test was used as a univariate analysis to compare the relapse-free survival (RFS) rates between UGVAB and SR groups. Cox regression analysis was used for multivariate analysis. RESULTS: A total of 39 patients were included. The median follow-up was 41 mo (range, 5-110 mo). The incidence of tumor recurrence was 23.1% (9/39). The 3-y cumulative RFS was 83.1% and 95.8% in the UGVAB and SR group, respectively, which was not significantly different between the two groups (P = 0.131, log-rank test). Multivariate analysis also revealed that treatment strategy (UGVAB versus SR) was not associated with an increased risk of relapse events (P = 0.274). CONCLUSIONS: Small desmoid tumors (≤3 cm) after UGVAB alone did not have a significantly compromised RFS compared with those who underwent SR. UGVAB may be an alternative and relatively conservative method for the diagnosis and local control of BDT with a smaller size. A prospective, randomized study with large sample size is needed to confirm this observation.

Pannexin 1 mediates ferroptosis that contributes to renal ischemia/reperfusion injury.

Renal ischemia/reperfusion injury (IRI) is a significant challenge in perioperative medicine and is related to oxidative programmed cell death. However, the role of ferroptosis, a newly discovered form of oxidative cell death, has not been evaluated widely. Pannexin 1 (PANX1), an ATP-releasing pathway family protein, has pro-apoptotic effects during kidney injury. Here, we demonstrate that PANX1 deletion protects against renal IRI by regulating ferroptotic cell death. Panx1 knockout mice subjected to renal IRI had decreased plasma creatinine, malondialdehyde (MDA) levels in kidney tissues, and tubular cell death (visible as decreased TUNEL-positive renal tubular cells) compared with WT mice. In cultured human kidney 2 (HK-2) cells, silenced Panx1 expression significantly attenuated ferroptotic lipid peroxidation and iron accumulation induced by the ferroptosis inducer erastin. Moreover, the Panx1 silencing significantly modulated ferroptosis-related protein expression. Furthermore, Panx1 deletion induced the expression of a cytoprotective chaperone, heme oxygenase-1 (HO-1), and inhibited ferroptinophagy via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway. In summary, Panx1 deletion protects against renal IRI by attenuating MAPK/ERK activation in a ferroptotic pathway. Our findings provide critical insights into the role of PANX1 in ferroptotic cell death and highlight a potential therapeutic target for the management of acute kidney injury (AKI) during the perioperative period.

Single Bimetallic Lanthanide-Based Metal-Organic Frameworks for Visual Decoding of a Broad Spectrum of Molecules.

Distinguishing the delicate structural differences among molecules is a critical and challenging task in biological/chemical analysis. A molecular decoding strategy has recently become promising to differentiate similar molecules, which is advantageous over the common sensing methods mostly used for detecting a single target. However, the design of an ideal molecular decoder is still strictly hindered by the tailored preparation of probes for particular molecules and the severe lack of widespread feasibility. We herein for the first time proposed to use single bimetallic lanthanide-based metal-organic frameworks (Ln-MOFs) as a powerful, versatile probe for fast and facile decoding of homologues, isomers, enantiomers, and even deuterated isotopomers, based on the unique host-guest interaction of a specific target with the Ln-MOF which could provide an according visual output based on the modulated energy transfer process.

Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes.

Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions, 192 novel protein start sites, 19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted "noncoding RNAs" to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes.