Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis.

Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available.ImportanceTo fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available

Construction of spirocarbocycles via gold-catalyzed intramolecular dearomatization of naphthols.

A highly efficient, gold-catalyzed intramolecular dearomatization reaction of naphthols via 5-endo-dig cyclization is described. This facile and direct approach furnishes spirocarbocycles in excellent yields under mild conditions

New class of 3D topological insulator in double perovskite

We predict a new class of three-dimensional topological insulators (TIs) in which the spin-orbit coupling (SOC) can more effectively generate a large band gap at $\Gamma$ point. The band gap of conventional TI such as Bi$_2$Se$_3$ is mainly limited by two factors, the strength of SOC and, from electronic structure perspective, the band gap when SOC is absent. While the former is an atomic property, we find that the latter can be minimized in a generic rock-salt lattice model in which a stable crossing of bands {\it at} the Fermi level along with band character inversion occurs for a range of parameters in the absence of SOC. Thus, large-gap TI's or TI's comprised of lighter elements can be expected. In fact, we find by performing first-principle calculations that the model applies to a class of double perovskites A$_2$BiXO$_6$ (A = Ca, Sr, Ba; X = Br, I) and the band gap is predicted up to 0.55 eV. Besides, more detailed calculations considering realistic surface structure indicate that the Dirac cones are robust against the presence of dangling bond at the boundary with a specific termination.Comment: submitted; title changed and new references added; see DOI for published versio

Seismic requalification of a safety class crane

A remotely operated 5-ton crane within a nuclear fuel handling facility was designed and constructed over 25 years ago. At that time, less severe design criteria, particularly on seismic loadings, were in use. This crane is being reactivated and requalified under new design criteria with loads including a site specific design basis earthquake. Detailed analyses of the crane show that the maximum stress coefficient is less than 90% of the code allowable, indicating that this existing crane is able to withstand loadings including those from the design basis earthquake. 3 refs., 8 figs., 2 tabs

Comparison of Sterile and Clean Dressing Techniques in Post-operative Surgical Wound Infection in a Chinese Healthcare Facility

Purpose: To investigate the effect of sterile and clean dressing techniques on wound management in a Chinese hospital, and to compare their impact on wound healing and the cost of the dressing materials with respect to postoperative surgical wounds.Methods: A total of 130 patients, comprising 70 (53.8 %) males and 60 (46.2 %) females, who had undergone surgery in The Affiliated Hospital of Changchun Traditional Chinese Medicine University, Changchun, China in 2012 – 2014 were enrolled in the study. Of these, 65 (50 %) received sterile dressings and 65 (50 %) clean dressings. A control group comprising 25 patients, 15 (60 %) males and 10 (40 %) females, who attended the clinic for change dressings only, was also included. The patients’ dressings were changed four times daily with 2x sterile and 2x clean dressings. Details of all the changes, including the nutritional status of the patients, were recorded. The patients were followed-up up to the time of their discharge.Results: Twelve (18.5 %) patients out of those who received sterile or clean dressings were found to have acquired an infection. The size of the wounds was approximately 1.8 to 32.4 cm3 (mean: 5.2 ± 6.4 cm3) in size at the start of the study and 0.6 to 4.2 cm3 at the end of the study. A significant difference was identified between the sterile and clean dressing groups at the beginning of the study (U = 72.5; p < 0.12). A decrease in wound size was observed in both of these groups but was not statistically significant, while the change in wound volume, was significantly different (U = 84.5; p < 0.25). When the cost of the two dressing types was compared, the sterile items were more expensive than that of the clean items; thus, sterile dressing procedure was significantly more costly than clean dressing procedure (p < 0.01).Conclusion: With mounting concern regarding antimicrobial resistance and hospital-acquired infections, suitable wound dressing techniques are required to prevent infection and reduce the duration of wound healing after surgery without compromising patient safety.Keywords: Wound dressing, Postoperative, Antimicrobial resistance, Hospital acquired infection

A comprehensive functional map of the hepatitis C virus genome provides a resource for probing viral proteins.

UnlabelledPairing high-throughput sequencing technologies with high-throughput mutagenesis enables genome-wide investigations of pathogenic organisms. Knowledge of the specific functions of protein domains encoded by the genome of the hepatitis C virus (HCV), a major human pathogen that contributes to liver disease worldwide, remains limited to insight from small-scale studies. To enhance the capabilities of HCV researchers, we have obtained a high-resolution functional map of the entire viral genome by combining transposon-based insertional mutagenesis with next-generation sequencing. We generated a library of 8,398 mutagenized HCV clones, each containing one 15-nucleotide sequence inserted at a unique genomic position. We passaged this library in hepatic cells, recovered virus pools, and simultaneously assayed the abundance of mutant viruses in each pool by next-generation sequencing. To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures. Moreover, we show the utility of these genetic footprints in the identification of candidate regions for epitope tag insertion. In a second application, we screened the genetic footprints for phenotypes that reflected defects in later steps of the viral life cycle. We confirmed that viruses with insertions in a region of the nonstructural protein NS4B had a defect in infectivity while maintaining genome replication. Overall, our genome-wide HCV mutant library and the genetic footprints obtained by high-resolution profiling represent valuable new resources for the research community that can direct the attention of investigators toward unidentified roles of individual protein domains.ImportanceOur insertional mutagenesis library provides a resource that illustrates the effects of relatively small insertions on local protein structure and HCV viability. We have also generated complementary resources, including a website (http://hangfei.bol.ucla.edu) and a panel of epitope-tagged mutant viruses that should enhance the research capabilities of investigators studying HCV. Researchers can now detect epitope-tagged viral proteins by established antibodies, which will allow biochemical studies of HCV proteins for which antibodies are not readily available. Furthermore, researchers can now quickly look up genotype-phenotype relationships and base further mechanistic studies on the residue-by-residue information from the functional profile. More broadly, this approach offers a general strategy for the systematic functional characterization of viruses on the genome scale

Impact of human papillomavirus vaccine on cervical cancer epidemic: Evidence from the surveillance, epidemiology, and end results program

IntroductionSince 2006, the human papillomavirus (HPV) vaccine has been recommended for females aged 9–26 years in the United States. Aiming to evaluate the early effect of the HPV vaccine on cervical cancer, this study assessed the incidence of cervical cancer by age and histology before and after the introduction of HPV vaccination.MethodsData on cervical cancer incidence from 1975–2019 were extracted from the Surveillance, Epidemiology, and End Results Program. Joinpoint regression was used to determine temporal trends over time. Future cervical cancer incidence (2015–2039) was projected using Bayesian age-period-cohort analysis. Age-period-cohort (APC) models were created to evaluate age, period, and cohort effects.ResultsFor overall cervical cancer and cervical squamous cell carcinoma (SCC), incidence rate showed decreasing trends (–0.7%, and –1.0% annually, respectively), whereas cervical adenocarcinoma (AC) incidence continuously increased (2.6% annually). The incidence trends for AC were stable in the 20–24 and 25–29-year age groups, whereas there was an increasing trend in older age groups. Similarly, the projected trend for AC in females aged 20–30 years exhibited a decline, whereas an increase was predicted in the 31–40–year age group, especially in the 35–44 year age group. The birth cohort and period effects in SCC and AC were extracted from APC models.DiscussionDuring the period of 1975–2019, the incidence of cervical AC remained almost unchanged in the age groups receiving HPV vaccines while increased in the age groups not receiving HPV vaccines. The birth cohort effects of SCC and AC of the cervix provided evidence supporting the effectiveness of the HPV vaccine in preventing cervical cancer