Directed Evolution of a Bacterial Leucyl tRNA in Mammalian Cells for Enhanced Noncanonical Amino Acid Mutagenesis.

The Escherichia coli leucyl-tRNA synthetase (EcLeuRS)/tRNAEcLeu pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNAEcLeu. Here, we overcome this limitation by evolving tRNAEcLeu directly in mammalian cells by using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNAEcLeu could be simultaneously engineered, which resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNAEcLeu, by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.

Enhanced Directed Evolution in Mammalian Cells Yields a Hyperefficient Pyrrolysyl tRNA for Noncanonical Amino Acid Mutagenesis.

Heterologous tRNAs used for noncanonical amino acid (ncAA) mutagenesis in mammalian cells typically show poor activity. We recently introduced a virus-assisted directed evolution strategy (VADER) that can enrich improved tRNA mutants from naïve libraries in mammalian cells. However, VADER was limited to processing only a few thousand mutants; the inability to screen a larger sequence space precluded the identification of highly active variants with distal synergistic mutations. Here, we report VADER2.0, which can process significantly larger mutant libraries. It also employs a novel library design, which maintains base-pairing between distant residues in the stem regions, allowing us to pack a higher density of functional mutants within a fixed sequence space. VADER2.0 enabled simultaneous engineering of the entire acceptor stem of M. mazei pyrrolysyl tRNA (tRNAPyl ), leading to a remarkably improved variant, which facilitates more efficient incorporation of a wider range of ncAAs, and enables facile development of viral vectors and stable cell-lines for ncAA mutagenesis.

Precise Manipulation of the Site and Stoichiometry of Capsid Modification Enables Optimization of Functional Adeno-Associated Virus Conjugates.

The ability to engineer adeno-associated virus (AAV) vectors for targeted transduction of specific cell types is critically important to fully harness their potential for human gene therapy. A promising approach to achieve this objective involves chemically attaching retargeting ligands onto the virus capsid. Site-specific incorporation of a bioorthogonal noncanonical amino acid (ncAA) into the AAV capsid proteins provides a particularly attractive strategy to introduce such modifications with exquisite precision. In this study, we show that using ncAA mutagenesis, it is possible to systematically alter the attachment site of a retargeting ligand (cyclic-RGD) on the AAV capsid to create diverse conjugate architectures and that the site of attachment heavily impacts the retargeting efficiency. We further demonstrate that the performance of these AAV conjugates is highly sensitive to the stoichiometry of capsid labeling (labels per capsid), with an intermediate labeling density providing optimal activity for cRGD-mediated retargeting. Finally, we developed a technology to more precisely control the number of attachment sites per AAV capsid by selectively incorporating an ncAA into the minor capsid proteins with high fidelity and efficiency, such that AAV conjugates with varying stoichiometry can be synthesized. Together, this platform provides unparalleled control over the site and stoichiometry of capsid modification, which will enable the development of next-generation AAV vectors tailored with desirable attributes.

Female Sexually Fluid (Non)identity Management Strategies at Work.

Previous research on sexual identity management at work focuses on default fixed sexual categories and related strategies such as pass or reveal. What if employees experience changes in their sexual attractions and identity? The present study pioneers in incorporating the concept of sexual fluidity into organization research in order to both complement and challenge the scholarship of sexual identity management at work. It provides empirical evidence from Vietnam that is in line with queer perspective in challenging the notion of a fixed, constructed sexual identity. Through in-depth interviews with 30 female employees experiencing sexual fluidity during their work, this study aims to investigate how they manage a sexually fluid identity in the workplace. The results show that female employees first consider or choose (non)identity1 that matches their new sexual attractions. These (non)identity choices include identity change, fluid identity, non-identity, and resisting identity. Next, strategies are utilized for managing that (non)identity at work-pass, cover, implicitly out, explicitly out, inform/educate. These strategies can be used independently or multiply (mixed/change), in which mixed strategy takes account of communication objects and situations, while change strategy relies on time. Associated individual, organizational and social factors are preliminarily mentioned, in which coworker reactions to the identity changes is a notable point (e.g., surprise, curiosity, inquiry, advice). This study also provides discussion on the construction of sexual identity as stigmatized social identity and the gap of scholarship of sexual identity management at work.

Virus-assisted directed evolution of biomolecules.

Directed evolution is a powerful technique that uses principles of natural evolution to enable the development of biomolecules with novel functions. However, the slow pace of natural evolution does not support the demand for rapidly generating new biomolecular functions in the laboratory. Viruses offer a unique path to design fast laboratory evolution experiments, owing to their innate ability to evolve much more rapidly than most living organisms, facilitated by a smaller genome size that tolerate a high frequency of mutations, as well as a fast rate of replication. These attributes offer a great opportunity to evolve various biomolecules by linking their activity to the replication of a suitable virus. This review highlights the recent advances in the application of virus-assisted directed evolution of designer biomolecules in both prokaryotic and eukaryotic cells.

Chemical Tagging of Bioactive Amides by Cooperative Catalysis: Applications in the Syntheses of Drug Conjugates.

We disclose a practical catalytic method for arming bioactive amide-based natural products and other small-molecule drugs with various functional handles for the synthesis of drug conjugates. We demonstrate that a set of readily available Sc-based Lewis acids and N-based Brønsted bases can function cooperatively to deprotonate amide N-H bonds in polyfunctional drug molecules. An aza-Michael reaction between the resulting amidate and α,ß-unsaturated compounds produces an array of drug analogues that are equipped with an alkyne, azide, maleimide, tetrazine, or diazirine moiety under redox and pH-neutral conditions. The utility of this chemical tagging strategy is showcased through the production of drug conjugates by the click reaction between the alkyne-tagged drug derivatives and an azide-containing green fluorescent protein, nanobody, or antibody.

Acceptability of prenatal diagnosis and prenatal treatment of haemophilia using cell and gene therapies within US haemophilia community.

BACKGROUND: The overall burden of disease in persons with haemophilia continues to be high despite the latest advancements in therapeutics. Clinical trials testing prenatal treatments for several genetic disorders are underway or are recruiting subjects, attesting to the much-needed change in paradigm of how patients with monogenic disorders can be treated. Here we investigate the overall attitude towards prenatal diagnosis, preferences on types of prenatal therapies for haemophilia, the level of 'acceptable' risk tolerated, and which social and moral pressures or disease personal experiences may predict willingness of individuals to consider foetal therapy in a future pregnancy. RESULTS: A multidisciplinary team designed the survey, and the study was carried out using REDCap, and publicized through the National Haemophilia Foundation. Subjects ≥18 years of age were eligible to participate in the study. We assessed participants' attitudes towards prenatal therapy and their level of 'acceptable' risk towards the procedure and therapy. The survey was completed by 67 adults, the majority females. Respondents were willing to undergo prenatal diagnosis, and their main concerns related to the well-being of the pregnant woman and the foetus regarding lasting therapeutic efficacy, side effects of the therapy, and procedural risks, but they were likely to accept a wide range of prenatal therapeutic options, particularly if the foetal therapy proved to be long-lasting and safe. CONCLUSIONS: These data demonstrate the willingness of persons with haemophilia, and the haemophilia community, to explore new treatment options beyond the currently offered approaches.

Photoredox-Catalyzed Labeling of Hydroxyindoles with Chemoselectivity (PhotoCLIC) for Site-Specific Protein Bioconjugation.

We have developed a novel visible-light-catalyzed bioconjugation reaction, PhotoCLIC, that enables chemoselective attachment of diverse aromatic amine reagents onto a site-specifically installed 5-hydroxytryptophan residue (5HTP) on full-length proteins of varied complexity. The reaction uses catalytic amounts of methylene blue and blue/red light-emitting diodes (455/650 nm) for rapid site-specific protein bioconjugation. Characterization of the PhotoCLIC product reveals a unique structure formed likely through a singlet oxygen-dependent modification of 5HTP. PhotoCLIC has a wide substrate scope and its compatibility with strain-promoted azide-alkyne click reaction, enables site-specific dual-labeling of a target protein.

Topologically associating domain boundaries are required for normal genome function.

Topologically associating domain (TAD) boundaries partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotypes1-3, but the overall extent to which this occurs remains unknown. Here we demonstrate that targeted deletions of TAD boundaries cause a range of disruptions to normal in vivo genome function and organismal development. We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11-80 kb in size) from the genome. All deletions examined resulted in detectable molecular or organismal phenotypes, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. We observed changes in local 3D chromatin architecture in 7 of 8 (88%) cases, including the merging of TADs and altered contact frequencies within TADs adjacent to the deleted boundary. For 5 of 8 (63%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and reinforce the critical need to carefully consider the potential pathogenicity of noncoding deletions affecting TAD boundaries in clinical genetics screening.

Pathogenic Role and Antibiotic Resistance of Methicillin-Resistant Staphylococcus aureus (MRSA) Strains Causing Severe Community-Acquired Pneumonia in Vietnamese Children.

In recent years, the pathogenic role and antibiotic resistance of methicillin-resistant Staphylococcus aureus (MRSA) strains causing severe community-acquired pneumonia (CAP) have received increasing attention in clinical practice. The aim of this study was to determine the rate of isolates of MRSA strains causing severe CAP in children and to assess their level of antibiotic resistance. The study design was cross-sectional. Children with severe CAP were sampled by nasopharyngeal aspiration for the culture, isolation, and identification of MRSA. Antimicrobial susceptibility testing was performed using the gradient diffusion method to determine the minimum inhibitory concentration (MIC) of antibiotics. Results: MRSA was identified as the second leading cause of severe CAP in Vietnamese children. The rate of isolates of S. aureus was 41/239 (17.5%), of which most were MRSA, at 32/41 (78.0%). MRSA strains were completely non-susceptible to penicillin (100%), more resistant to clindamycin and erythromycin, less sensitive to ciprofloxacin and levofloxacin, and fully susceptible to vancomycin and linezolid, with a 32-fold decreased MIC90 for vancomycin (0.5 mg/L) and a 2-fold decreased MIC90 for linezolid (4 mg/L). Therefore, vancomycin and linezolid may be appropriate options for severe CAP identified by MRSA.

Thymic stromal lymphopoietin-stimulated CD4+ T cells induce senescence in advanced breast cancer.

Thymic Stromal Lymphopoietin (TSLP) plays a prominent role in inducing type 2 immune response, commonly associated with atopic diseases. TSLP-activated CD4+ T helper 2 cells block early carcinogenesis by inducing terminal differentiation in spontaneous breast and lung cancer models. However, the impact of TSLP induction on advanced cancer with altered cellular phenotypes is unclear. Using an established MMTV-PyMttg breast cancer cell line, we demonstrate that TSLP-stimulated CD4+ T cells possess an antitumor effect in advanced breast cancer. In contrast to early breast cancer suppression, the antitumor immunity mediated by TSLP-stimulated CD4+ T cells in advanced breast cancer is mediated by the induction of a senescent-like phenotype in cancer cells. Inflammatory CD4+ T cells drive breast cancer cells into senescence by releasing interferon-gamma and tumor necrosis factor-alpha, which directly bind to their receptors on cancer cells. Our findings reveal a novel mechanism of TSLP-activated CD4+ T cell immunity against advanced breast cancer, mediated by cellular senescence as a distinct effector mechanism for cancer immunotherapy.

Genome-wide fetalization of enhancer architecture in heart disease.

Heart disease is associated with re-expression of key transcription factors normally active only during prenatal development of the heart. However, the impact of this reactivation on the regulatory landscape in heart disease is unclear. Here, we use RNA-seq and ChIP-seq targeting a histone modification associated with active transcriptional enhancers to generate genome-wide enhancer maps from left ventricle tissue from up to 26 healthy controls, 18 individuals with idiopathic dilated cardiomyopathy (DCM), and five fetal hearts. Healthy individuals have a highly reproducible epigenomic landscape, consisting of more than 33,000 predicted heart enhancers. In contrast, we observe reproducible disease-associated changes in activity at 6,850 predicted heart enhancers. Combined analysis of adult and fetal samples reveals that the heart disease epigenome and transcriptome both acquire fetal-like characteristics, with 3,400 individual enhancers sharing fetal regulatory properties. We also provide a comprehensive data resource (http://heart.lbl.gov) for the mechanistic exploration of DCM etiology.

The effectiveness of Paraffin oil and Mineral oil for day-5 embryo culture in couples undergoing in vitro fertilisation.

OBJECTIVE: This study evaluated the effectiveness of Paraffin oil versus Mineral oil for day-5 embryo culture in couples undergoing assisted reproductive technology (ART). METHODS: We performed a multi-centre, retrospective cohort study at IVFMD (My Duc Hospital) and IVFMD Phu Nhuan (My Duc Phu Nhuan Hospital) from January 2019 to September 2019. We studied couples treated by intracytoplasmic sperm injection (ICSI), using fresh, ejaculated semen and undergoing day-5 embryo transfer. Couples who underwent in vitro maturation (IVM) or oocyte donation cycles or couples where the woman had uterine abnormalities were excluded. From January 2019 to May 2019, we used Mineral oil (LiteOil, LifeGlobal) while Paraffin oil (Liquid Paraffin, Origio) was used from June 2019 to September 2019. The primary outcome was live birth rate after the first transfer, either from a fresh transfer or frozen embryo transfer. RESULTS: Between 1st January 2019 to 30th September 2019, there were 2,312 couples undergoing ART in both centres, of which 762 (377 in the Paraffin group and 385 in the Mineral group) eligible couples were included in the study. Baseline characteristics of couples were comparable between the two groups, with mean female age 31.5 ± 4.3 versus 31.9 ± 4.7 in the Paraffin and Mineral group. Live birth after the first transfer occurred in 153 (40.6%) couples in the Paraffin group, compared to 152 (39.5%) couples in the Mineral group (risk ratio 1.02, 95% confidence interval 0.91 - 1.14). Other secondary outcomes were comparable between the two groups. CONCLUSION: In day-5 embryo culture, Paraffin and Mineral oil resulted in a comparable live birth rate.

Investigating the microbial pathogens of sexually transmitted infections among heterosexual Vietnamese men with symptomatic urethritis.

OBJECTIVE: To explore the microbial etiology of urethritis in Vietnamese men and the association with patients' characteristics, especially their sexual behaviors. METHODS: This study was conducted on 349 men who presented with symptomatic urethritis and evidence of STIs (determined by multiplex PCR tests) at the Department of Andrology and Sexual Medicine-Hanoi Medical University Hospital. All information regarding medical history, sexual activities, and symptoms of urethritis was documented. RESULTS: C. trachomatis and N. gonorrhoea remained the two most common causative pathogens, followed by an unexpectedly high prevalence of Mycoplasma and Ureaplasma species. Coinfection was significant with a rate of 40.7%. Men who had sex with female sex workers (FSWs) were more likely to be positive with N. gonorrhoea but less likely to be positive with C. trachomatis and M. genitalium than those having sex with only one romantic partner. CONCLUSIONS: Our findings suggested the important role of other microorganisms, especially M. genitalium, in the etiology of urethritis in men besides the previously well-known causes of STIs. Since the coinfection rate is quite high, targeted treatment with clear microbial evidence should be considered rather than empiric antimicrobial therapy.

Target Design of Novel Histone Deacetylase 6 Selective Inhibitors with 2-Mercaptoquinazolinone as the Cap Moiety.

Epigenetic alterations found in all human cancers are promising targets for anticancer therapy. In this sense, histone deacetylase inhibitors (HDACIs) are interesting anticancer agents that play an important role in the epigenetic regulation of cancer cells. Here, we report 15 novel hydroxamic acid-based histone deacetylase inhibitors with quinazolinone core structures. Five compounds exhibited antiproliferative activity with IC50 values of 3.4-37.8 µM. Compound 8 with a 2-mercaptoquinazolinone cap moiety displayed the highest antiproliferative efficacy against MCF-7 cells. For the HDAC6 target selectivity study, compound 8 displayed an IC50 value of 2.3 µM, which is 29.3 times higher than those of HDAC3, HDAC4, HDAC8, and HDAC11. Western blot assay proved that compound 8 strongly inhibited tubulin acetylation, a substrate of HDAC6. Compound 8 also displayed stronger inhibition activity against HDAC11 than the control drug Belinostat. The inhibitory mechanism of action of compound 8 on HDAC enzymes was then explored using molecular docking study. The data revealed a high binding affinity (-7.92 kcal/mol) of compound 8 toward HDAC6. In addition, dock pose analysis also proved that compound 8 might serve as a potent inhibitor of HDAC11.

Factors Associated with Community Awareness on COVID-19 in a Developing Country: Implications for Optimal Risk Communication.

Purpose: New COVID-19 variants pose great challenges in protecting the success of vaccination programs. Awareness of community becomes an important component to prevent the spread of COVID-19. This study aims to analyze people's awareness of COVID-19 and identify the implications for optimal risk communication in Vietnam. Study Methods and Materials: A total of 341 individuals participated in an online cross-sectional study. Community awareness on COVID-19 was examined by using a structured questionnaire. Multivariable Tobit regression was used to examine associated factors with community awareness. Results: Our results indicated high levels of awareness of local COVID-19 situation, transmission risks, and preventive behavioral practices during the first week of social isolation. Higher awareness of local pandemic situation was associated with female respondents (Coef.: 6.19; 95% CI: 0.51; 11.87) and larger family sizes of above 5 people (Coef.: 9.00; -1.00; 19.00). Respondents between 35-44 years old were shown to be less aware of preventive behavioral practices than other age groups, including the group of participants above 44 years old (Coef.: -0.34; 95% CI: -0.67; -0.02). Lastly, participants who were "fairly satisfied" with information resources had awareness levels of preventive behavioral practices lower than that of "unsatisfied" respondents (Coef.: -0.45; 95% CI: 0.74; -0.16). Conclusion: This study offers crucial insights into the pandemic awareness of citizens and risk communication effectiveness during COVID-19 in Vietnam. The study findings shall serve the development of policies and interventions aimed at empowering individuals in the local and global fight against coronavirus.

Knowledge and Self-Protective Practices Against COVID-19 Among Healthcare Workers in Vietnam.

Background: In middle-income countries such as Vietnam, where healthcare resources are already constrained, protecting healthcare workers (HCWs) is essential for ensuring the sustainability of COVID-19 response in Vietnam. This study was conducted to assess the knowledge and practices regarding the prevention of the COVID-19 among the HCWs in Vietnam to identify the ways of disseminating information to maximize the safety of these essential workers. Methods: An online cross-sectional study, using respondent-driven sampling, was conducted in Vietnam with 742 participants within 2 weeks. The validity of the questionnaire was examined by exploratory factor analysis. Descriptive statistics were used to identify the level of knowledge and practices among the HCWs to prevent the COVID-19. Inferential statistics and regression modeling were used to identify the associated factors with results. Results: Vietnamese HCWs had a high level of knowledge with more than 75% of the participants demonstrating awareness of all the modes of transmission aside from air. The mean knowledge score was 3.7 ± 0.8 (range 1-5). Nearly all the participants relied on the Ministry of Health (98.3%) and the internet (95.5%) for information regarding the COVID-19. The participants endorsed a moderately high level of self-protective practices with mean scores of 4.2 and 3.6 (band score 1-5) for the precautionary and psychological measures, respectively. Nurses were more likely to practice the precautionary measures than doctors and the HCWs at the central level were more likely to practice the psychological measures than those at the district level. Conclusion: Future education initiatives should consolidate the latest literature in an accessible format, focusing initially on the gaps of knowledge regarding aerosol transmission. These initiatives should primarily focus on the doctors, especially those in emergency and intensive care departments.

Impacts of COVID-19 on the Life and Work of Healthcare Workers During the Nationwide Partial Lockdown in Vietnam.

Background: Healthcare workers are frontline responders facing a disproportionate increase in occupational responsibilities during the COVID-19 pandemic. Added work-related stress among healthcare personnel may lead to personal and work-related repercussions, such as burnout or decreased quality of care for patients; however, little is known about how the COVID-19 pandemic affects the daily work and life of these workers. This study aimed to evaluate the personal and occupational impacts of the COVID-19 induced partial lockdown in Vietnam among hospital staff. Methods: A cross-sectional web-based study was carried out to collect demographic data and the personal and job impacts of respondents during the second week of national lockdown in April 2020. Snowball sampling technique was applied to recruit 742 hospital staff. The exploratory factor analysis (EFA) was used to examine the validity of the instrument. Results: Of the 742 respondents, 21.2% agreed that "working attitude well-maintained," followed by 16.1% of respondents who reported that there were "enough employees at work." Only 3.2% of respondents agreed that "their work was appreciated by society." Furthermore, healthcare workers in the central region were less likely to have experienced "Avoidance of disclosure and discrimination related to COVID-19" than other areas (Coef. = - 0.25, CI: -0.42 to -0.07). Being women also had a negative association with scores in "Avoidance of disclosure and discrimination related to COVID 19" domain (Coef. = -0.27, CI: -0.43 to -0.12) while having a positive association with "negative attitude towards working conditions" domain (Coef. = 0.19, CI: 0.09 to 0.3). In addition, working in administrative offices (Coef. = 0.20; 95% CI = 0.05 to 0.36) and infectious departments (Coef. = 0.36; 95% CI = 0.09 to 0.63) had a positive association with "Increased work pressure due to COVID 19" domain. Conclusion: These findings revealed marginal impacts of the COVID-19 pandemic on the work and life of hospital staff in Vietnam. Furthermore, this study highlighted the importance of implementing preventive strategies during the nationwide partial lockdown to manage hospital admissions and the burden on healthcare workers. Finally, this study characterizes targeted demographics that may benefit from appreciation by employers and society during a national pandemic.

Psychosocial Impacts of COVID-19 on Healthcare Workers During the Nationwide Partial Lockdown in Vietnam in April 2020.

Background: The psychosocial impact of COVID-19 is greater among healthcare workers (HCWs) than the general population. This study aims to identify psychosocial problems faced by HCWs in Vietnam during the national partial lockdown between 1 and 22 April 2020 and to identify risk factors associated with psychosocial issues among this population. Methods: A cross-sectional study was conducted in the second week of April 2020 during the national lockdown in Vietnam. Snowball sampling technique was used to recruit participants through web-based surveys. The Impact of Events Scale-Revised (IES-R) was used to assess the impact of COVID-19 on HCWs through online surveys. Results: Of the 349 HCWs, we found 22.6% reported psychosocial problems. Most of participants reported having exposure to COVID-19 daily (48.7%). The majority of them also felt that their job put them at risk of SARS-CoV-2 infections (90.3%) and expressed fear of potential infection (85.7%). Despite COVID-19 risks, 95.4% of participants, however, expressed their willingness to continue working at their current health facility. In addition, 94.8% of participants believed if they or their family members had been infected, their agency leaders would have provided them with appropriate medical care. Lastly, HCWs who worked in the internal medicine department who did not take care of COVID-19 patients or expressed fear of becoming infected were more likely to have higher total IES-R scores. Conclusion: Our findings suggest that the support of healthcare leaders and assurance of care might be helpful in mitigating the psychological effects of COVID-19 among HCWs in Vietnam. These resources should be tailored to HCWs who are working in different areas of health services, including staff who are not working directly with COVID-19 patients. In addition, psychosocial health resources should be provided for not only physicians but also nursing staff.