Proximity labeling of host factor ANXA3 in HCV infection reveals a novel LARP1 function in viral entry.

Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments, ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.

Efficient clathrin-mediated entry of enteric adenoviruses in human duodenal cells.

IMPORTANCE: Enteric adenoviruses have historically been difficult to grow in cell culture, which has resulted in lack of knowledge of host factors and pathways required for infection of these medically relevant viruses. Previous studies in non-intestinal cell lines showed slow infection kinetics and generated comparatively low virus yields compared to other adenovirus types. We suggest duodenum-derived HuTu80 cells as a superior cell line for studies to complement efforts using complex intestinal tissue models. We show that viral host cell factors required for virus entry differ between cell lines from distinct origins and demonstrate the importance of clathrin-mediated endocytosis.

We Are Not Speaking the Same Language: Current Procedural Terminology Coding and Provision of Care in Lymphatic Reconstruction.

INTRODUCTION: Current Procedural Terminology (CPT) codes provide a uniform language for medical billing, but specific codes have not been assigned for lymphatic reconstruction techniques. The authors hypothesized that inadequate codes would contribute to heterogeneous coding practices and reimbursement challenges, ultimately limiting surgeons' ability to treat patients. METHODS: A 22-item virtual questionnaire was offered to 959 members of the American Society of Reconstructive Microsurgeons to assess the volume of lymphatic reconstruction procedures performed, CPT codes used for each procedure, and challenges related to coding and providing care. RESULTS: The survey was completed by 66 board-certified/board-eligible plastic surgeons (6.9%), who unanimously agreed that lymphatic surgery is integral to cancer care, with 86.4% indicating that immediate lymphatic reconstruction should be offered after lymphadenectomy. Most performed lymphovenous bypass, immediate lymphatic reconstruction, liposuction, and vascularized lymph node transfer.Respondents reported that available CPT codes failed to reflect procedural scope. A wide variety of CPT codes was used to report each type of procedure. Insurance coverage problems led to 69.7% of respondents forgoing operations and 32% reducing treatment offerings. Insurance coverage and CPT codes were identified as significant barriers to care by 98.5% and 95.5% of respondents, respectively. CONCLUSIONS: Respondents unanimously agreed on the importance of lymphatic reconstruction in cancer care, and most identified inadequate CPT codes as causing billing issues, which hindered their ability to offer surgical treatment. Appropriate and specific CPT codes are necessary to ensure accuracy and consistency of reporting and ultimately to improve patient access to care.

Geospatial and Socioeconomic Disparities Influencing the Management of Craniosynostosis.

BACKGROUND: Various social determinants of health have been described as predictors of clinical outcomes for the craniosynostosis population. However, literature lacks a granular depiction of socioeconomic factors that impact these outcomes, and little is known about the relationship between patients' proximity to the care center and management of the condition. METHODS/DESIGN: This study retrospectively evaluated patients with craniosynostosis who presented to a tertiary children's hospital between 2000 and 2019. Outcomes of interest included age at presentation for surgery, incidence of reoperation, and length of follow-up. Patient addresses were geocoded and plotted on two separate shapefiles containing block group information within San Diego County. The shapefiles included percent parental educational attainment (bachelor's degree or higher) and median household income from 2010. The year 2010 was chosen for the shapefiles because it is the median year of data collection for this study. Multivariate linear, logistic, and polynomial regression models were used to analyze the relationship between geospatial and socioeconomic predictors and clinical outcomes. RESULTS: There were 574 patients with craniosynostosis included in this study. The mean ± SD Haversine distance from the patient's home coordinates to the hospital coordinates was 107.2 ± 321.2 miles. After adjusting for the suture fused and insurance coverage, there was a significant positive correlation between distance to the hospital and age at index surgery (P = 0.018). There was no correlation between distance and incidence of reoperation (P = 0.266) or distance and duration of follow-up (P = 0.369). Using the same statistical adjustments, lower parental percent educational attainment and lower median household income correlated with older age at index surgery (P = 0.008 and P = 0.0066, respectively) but were not correlated with reoperation (P = 0.986 and P = 0.813, respectively) or duration of follow-up (P = 0.107 and P = 0.984, respectively). CONCLUSIONS: The results offer evidence that living a greater distance from the hospital and socioeconomic disparities including parental education and median household income may serve as barriers to prompt recognition of diagnosis and timely care in this population. However, the geospatial and socioeconomic factors studied do not seem to hinder incidence of reoperation or length of follow-up, suggesting that, once care has been initiated, longitudinal outcomes may be less impacted.

Geospatial and Socioeconomic Disparities Influencing the Management and Outcomes of Nonsyndromic Craniosynostosis: A Systematic Review.

Geospatial and socioeconomic health disparities are potential barriers to timely diagnosis and treatment of nonsyndromic craniosynostosis. This systematic review aims to assess published literature describing disparities in craniosynostosis care and to summarize the findings surrounding patient proximity to care centers and familial socioeconomic status as predictors of surgical management and outcomes. The data sources used include PubMed, MEDLINE, and Google Scholar. The Strengthening the Reporting of Observational Studies in Epidemiology checklist was used for appraisal of the quality of the studies included. Generally, the literature reviewed suggested that socioeconomic variables including race, insurance payor, and median zip code income quartile are predictors of suboptimal craniosynostosis surgical management outcomes including older age at time of surgery and more invasive surgical approach performed. The only geospatial data element assessed was the general region of the hospital where the patient was treated. The review highlighted various knowledge gaps within published literature describing health-related disparities in patients with craniosynostosis. There is a paucity of research assessing geospatial access to craniosynostosis care centers, suggesting that further research should be performed to evaluate this potential disparity. In addition, previous studies lack granularity when assessing socioeconomic factors and only one study accounted for suture fused, which is a potential confounding variable across the other published work. These considerations should be addressed in future studies addressing this topic. The limitations of this review include potential publication bias given that unpublished work was not included. An element of reviewer bias also exists considering only one reviewer screened the articles and extracted the data.

A Work in Progress: Women's Status in the Plastic Surgery Workforce and Recommendations for Success.

BACKGROUND: Only 20% of the current plastic surgery workforce is female, but since 2022, most matched applicants in integrated plastic surgery programs have been women. This study aimed to examine current practice models among female plastic surgeons, including those outside of academia, as the field continues to evolve. OBJECTIVES: This study surveyed female plastic surgeons in different practice models to elicit perspectives, career path advice, characterize/project trends, and provide recommendations for success. METHODS: A 37-question survey focusing on demographics, practice models, career paths, desire for practice changes and advice for women trainees was emailed to 1342 members of the American Society of Plastic Surgeons and The Aesthetic Society. Chi square analyses compared practice type characteristics (academia, employed roles, private practice), at a significance level of 0.05. RESULTS: Response rate was 53%. Most respondents were age 35-45, in solo practice, trained via traditional pathways, without fellowships, and lacked female mentors. Private practice surgeons were very satisfied in their career, employed surgeons were moderately satisfied and academic surgeons were mildly satisfied. Academic surgeons reported a higher number of working hours (>60) and cases per month (20-30) than employed or private practice surgeons. Recommendations for success included seeking female mentorship, enhancing business skills and building a social media platform during training. CONCLUSIONS: Results highlight the importance of female mentorship, acquiring business skills, and indicate private practice leads to greater career satisfaction. Training programs should consider accounting for these factors to better promote women's success and improve equity in academic practice.

Luminescent reporter cells enable the identification of broad-spectrum antivirals against emerging viruses.

The emerging viruses SARS-CoV-2 and arenaviruses cause severe respiratory and hemorrhagic diseases, respectively. The production of infectious particles of both viruses and virus spread in tissues requires cleavage of surface glycoproteins (GPs) by host proprotein convertases (PCs). SARS-CoV-2 and arenaviruses rely on GP cleavage by PCs furin and subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P), respectively. We report improved luciferase-based reporter cell lines, named luminescent inducible proprotein convertase reporter cells that we employ to monitor PC activity in its authentic subcellular compartment. Using these sensor lines we screened a small compound library in high-throughput manner. We identified 23 FDA-approved small molecules, among them monensin which displayed broad activity against furin and SKI-1/S1P. Monensin inhibited arenaviruses and SARS-CoV-2 in a dose-dependent manner. We observed a strong reduction in infectious particle release upon monensin treatment with little effect on released genome copies. This was reflected by inhibition of SARS-CoV-2 spike processing suggesting the release of immature particles. In a proof of concept experiment using human precision cut lung slices, monensin potently inhibited SARS-CoV-2 infection, evidenced by reduced infectious particle release. We propose that our PC sensor pipeline is a suitable tool to identify broad-spectrum antivirals with therapeutic potential to combat current and future emerging viruses.

Nuclear PYHIN proteins target the host transcription factor Sp1 thereby restricting HIV-1 in human macrophages and CD4+ T cells.

Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity.

Delivering the unexpected-Information needs for PSA screening from Men's perspective: A qualitative study.

BACKGROUND: Making decisions about PSA screening tests is challenging, as it requires both knowledge of the possible benefits and harms of screening and an individual assessment of the patient's values. Our research explores how much and what information men perceive to be necessary with regard to screening for prostate cancer. OBJECTIVE: To explore men's information and associated needs for decision making in PSA testing. DESIGN: Qualitative interview study. SETTING AND PARTICIPANTS: We interviewed 32 men (aged 55-69) about their decision making on PSA screening following counselling with a Decision Aid at their GP's or urologist's practice in Germany. MAIN OUTCOME MEASURES: Men's expressed needs for decision making in PSA testing. METHODS: All interviews were transcribed verbatim and analysed by framework analysis. RESULTS: Comprehensive pre-screening counselling is needed. For the men in our study, information about test (in)accuracy, the benefit-harm balance and consequences of the test were relevant and surprising. Additional needs were for interpretation support, a take-home summary and time for deliberation. For several men, their physician's attitude was of interest. After being well-informed, most men felt empowered to make a preference-based decision on their own. DISCUSSION: Men were surprised by what they learned, especially regarding the accuracy and possible harms of screening. There is large variation in the breadth and depth of information needed, and some controversy regarding the consequences of testing. CONCLUSION AND PATIENT CONTRIBUTION: A core set of information should be offered before men make their first PSA screening decision. Information about biopsy and associated side-effects could follow in a short form, with details only on request. Knowledge about a high rate of false-positive test results beforehand might help men handle a suspicious test result.

Prophylactic Antiviral Activity of Sulfated Glycomimetic Oligomers and Polymers.

In this work, we investigate the potential of highly sulfated synthetic glycomimetics to act as inhibitors of viral binding/infection. Our results indicate that both long-chain glycopolymers and short-chain glycooligomers are capable of preventing viral infection. Notably, glycopolymers efficiently inhibit Human Papillomavirus (HPV16) infection in vitro and maintain their antiviral activity in vivo, while the glycooligomers exert their inhibitory function post attachment of viruses to cells. Moreover, when we tested the potential for broader activity against several other human pathogenic viruses, we observed broad-spectrum antiviral activity of these compounds beyond our initial assumptions. While the compounds tested displayed a range of antiviral efficacies, viruses with rather diverse glycan specificities such as Herpes Simplex Virus (HSV), Influenza A Virus (IAV), and Merkel Cell Polyomavirus (MCPyV) could be targeted. This opens new opportunities to develop broadly active glycomimetic inhibitors of viral entry and infection.

Infectious Entry of Merkel Cell Polyomavirus.

Merkel cell polyomavirus (MCPyV) is a small, nonenveloped tumor virus associated with an aggressive form of skin cancer, Merkel cell carcinoma (MCC). MCPyV infections are highly prevalent in the human population, with MCPyV virions being continuously shed from human skin. However, the precise host cell tropism(s) of MCPyV remains unclear: MCPyV is able to replicate within a subset of dermal fibroblasts, but MCPyV DNA has also been detected in a variety of other tissues. However, MCPyV appears different from other polyomaviruses, as it requires sulfated polysaccharides, such as heparan sulfates and/or chondroitin sulfates, for initial attachment. Like other polyomaviruses, MCPyV engages sialic acid as a (co)receptor. To explore the infectious entry process of MCPyV, we analyzed the cell biological determinants of MCPyV entry into A549 cells, a highly transducible lung carcinoma cell line, in comparison to well-studied simian virus 40 and a number of other viruses. Our results indicate that MCPyV enters cells via caveolar/lipid raft-mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis, or glycosphingolipid-enriched carriers. The viruses were internalized in small endocytic pits that led the virus to endosomes and from there to the endoplasmic reticulum (ER). Similar to other polyomaviruses, trafficking required microtubular transport, acidification of endosomes, and a functional redox environment. To our surprise, the virus was found to acquire a membrane envelope within endosomes, a phenomenon not reported for other viruses. Only minor amounts of viruses reached the ER, while the majority was retained in endosomal compartments, suggesting that endosome-to-ER trafficking is a bottleneck during infectious entry.IMPORTANCE MCPyV is the first polyomavirus directly implicated in the development of an aggressive human cancer, Merkel cell carcinoma (MCC). Although MCPyV is constantly shed from healthy skin, the MCC incidence increases among aging and immunocompromised individuals. To date, the events connecting initial MCPyV infection and subsequent transformation still remain elusive. MCPyV differs from other known polyomaviruses concerning its cell tropism, entry receptor requirements, and infection kinetics. In this study, we examined the cellular requirements for endocytic entry as well as the subcellular localization of incoming virus particles. A thorough understanding of the determinants of the infectious entry pathway and the specific biological niche will benefit prevention of virus-derived cancers such as MCC.

Extracellular Conformational Changes in the Capsid of Human Papillomaviruses Contribute to Asynchronous Uptake into Host Cells.

Human papillomavirus 16 (HPV16) is the leading cause of cervical cancer. For initial infection, HPV16 utilizes a novel endocytic pathway for host cell entry. Unique among viruses, uptake occurs asynchronously over a protracted period of time, with half-times between 9 and 12 h. To trigger endocytic uptake, the virus particles need to undergo a series of structural modifications after initial binding to heparan sulfate proteoglycans (HSPGs). These changes involve proteolytic cleavage of the major capsid protein L1 by kallikrein-8 (KLK8), exposure of the N terminus of the minor capsid protein L2 by cyclophilins, and cleavage of this N terminus by furin. Overall, the structural changes are thought to facilitate the engagement of an elusive secondary receptor for internalization. Here, we addressed whether structural changes are the rate-limiting steps during infectious internalization of HPV16 by using structurally primed HPV16 particles. Our findings indicate that the structural modifications mediated by cyclophilins and furin, which lead to exposure and cleavage, respectively, of the L2 N terminus contribute to the slow and asynchronous internalization kinetics, whereas conformational changes elicited by HSPG binding and KLK8 cleavage did not. However, these structural modifications accounted for only 30 to 50% of the delay in internalization. Therefore, we propose that limited internalization receptor availability for engagement of HPV16 causes slow and asynchronous internalization in addition to rate-limiting structural changes in the viral capsid.IMPORTANCE HPVs are the main cause of anogenital cancers. Their unique biology is linked to the differentiation program of skin or mucosa. Here, we analyzed another unique aspect of HPV infections using the prototype HPV16. After initial cell binding, HPVs display an unusually protracted residence time on the plasma membrane prior to asynchronous uptake. As viruses typically do not expose themselves to host immune sensing, we analyzed the underlying reasons for this unusual behavior. This study provides evidence that both extracellular structural modifications and possibly a limited availability of the internalization receptor contribute to the slow internalization process of the virus. These findings indicate that perhaps a unique niche for initial infection that could allow for rapid infection exists. In addition, our results may help to develop novel, preventive antiviral measures.

A central region in the minor capsid protein of papillomaviruses facilitates viral genome tethering and membrane penetration for mitotic nuclear entry.

Incoming papillomaviruses (PVs) depend on mitotic nuclear envelope breakdown to gain initial access to the nucleus for viral transcription and replication. In our previous work, we hypothesized that the minor capsid protein L2 of PVs tethers the incoming vDNA to mitotic chromosomes to direct them into the nascent nuclei. To re-evaluate how dynamic L2 recruitment to cellular chromosomes occurs specifically during prometaphase, we developed a quantitative, microscopy-based assay for measuring the degree of chromosome recruitment of L2-EGFP. Analyzing various HPV16 L2 truncation-mutants revealed a central chromosome-binding region (CBR) of 147 amino acids that confers binding to mitotic chromosomes. Specific mutations of conserved motifs (IVAL286AAAA, RR302/5AA, and RTR313EEE) within the CBR interfered with chromosomal binding. Moreover, assembly-competent HPV16 containing the chromosome-binding deficient L2(RTR313EEE) or L2(IVAL286AAAA) were inhibited for infection despite their ability to be transported to intracellular compartments. Since vDNA and L2 were not associated with mitotic chromosomes either, the infectivity was likely impaired by a defect in tethering of the vDNA to mitotic chromosomes. However, L2 mutations that abrogated chromatin association also compromised translocation of L2 across membranes of intracellular organelles. Thus, chromatin recruitment of L2 may in itself be a requirement for successful penetration of the limiting membrane thereby linking both processes mechanistically. Furthermore, we demonstrate that the association of L2 with mitotic chromosomes is conserved among the alpha, beta, gamma, and iota genera of Papillomaviridae. However, different binding patterns point to a certain variance amongst the different genera. Overall, our data suggest a common strategy among various PVs, in which a central region of L2 mediates tethering of vDNA to mitotic chromosomes during cell division thereby coordinating membrane translocation and delivery to daughter nuclei.

Large scale RNAi reveals the requirement of nuclear envelope breakdown for nuclear import of human papillomaviruses.

A two-step, high-throughput RNAi silencing screen was used to identify host cell factors required during human papillomavirus type 16 (HPV16) infection. Analysis of validated hits implicated a cluster of mitotic genes and revealed a previously undetermined mechanism for import of the viral DNA (vDNA) into the nucleus. In interphase cells, viruses were endocytosed, routed to the perinuclear area, and uncoated, but the vDNA failed to be imported into the nucleus. Upon nuclear envelope perforation in interphase cells HPV16 infection occured. During mitosis, the vDNA and L2 associated with host cell chromatin on the metaphase plate. Hence, we propose that HPV16 requires nuclear envelope breakdown during mitosis for access of the vDNA to the nucleoplasm. The results accentuate the value of genes found by RNAi screens for investigation of viral infections. The list of cell functions required during HPV16 infection will, moreover, provide a resource for future virus-host cell interaction studies.

Maximizing Patient Satisfaction in Facial Feminizing Rhinoplasty.

Background: Although facial feminizing rhinoplasty can reduce gender dysphoria, there is limited evidence on approaches to maximize transgender patient satisfaction. In a retrospective cohort of transfeminine patients who underwent feminizing rhinoplasty, we compare pre- and postoperative nasal metrics and postoperative satisfaction. Methods: Records were retrospectively reviewed to identify transfeminine patients who had feminizing rhinoplasty and cisgender females who had aesthetic rhinoplasty at least 8 weeks post-rhinoplasty. Transgender patients were contacted to rate their aesthetic and functional rhinoplasty satisfaction. Patients with 75% or greater of the total survey score were "very satisfied," those between 50% and 75% were "satisfied," and those below 50% were "less satisfied." The Vectra 3D imaging software was utilized to measure each patient's pre- and post-rhinoplasty dorsal lengths; tip projection ratios; and nasolabial, nasofrontal, and nasofacial angles. Relative percent changes for each patient between pre- and post-rhinoplasty measurements were compared between transgender and cisgender females using descriptive statistics. Results: Twenty-five transgender patients met the inclusion criteria; 19 answered the survey with 12 very satisfied, 7 satisfied, and 0 less satisfied patients. The median age of surveyed patients was 35, and 42.1% identified as Hispanic. Between very satisfied and satisfied patients, median relative percent changes in dorsal length (-1.2% vs 5.7%, P = .043), tip projection ratio (2.4% vs 8.1%, P = .038), and nasolabial angle (-2.5% vs 9.7%, P = .026) significantly differed; median relative changes in nasofrontal angles (4.2% vs -0.6%, P = .071) and nasofacial angles (-0.7% vs -3.6%, P = .703) were insignificantly different. Satisfied transgender patients and cisgender patients (n = 5) had significant differences in median relative changes in dorsal length (5.7% vs 0.7%, P = .047), tip projection ratio (8.1% vs -3.5%, P = .033), and nasolabial angles (9.7% vs -5.4%, P = .042). Very satisfied transgender and cisgender females had no significant differences in relative metric changes. Conclusions: Very satisfied transgender patients had decreases in dorsal length, smaller increases in tip projection ratio, and decreases in the nasolabial angle compared with satisfied patients. These data can help focus feminizing rhinoplasty approaches to maximize satisfaction. Further, very satisfied transgender patients had similar changes as cisgender females, reaffirming the utility of applying cisgender female rhinoplasty considerations to feminizing rhinoplasty.

The blue light-dependent LOV-protein LdaP of Dinoroseobacter shibae acts as antirepressor of the PpsR repressor, regulating photosynthetic gene cluster expression.

In the marine α-proteobacterium Dinoroseobacter shibae more than 40 genes of the aerobic anoxygenic photosynthesis are regulated in a light-dependent manner. A genome-wide screen of 5,605 clones from a D. shibae transposon library for loss of pigmentation and changes in bacteriochlorophyll absorbance identified 179 mutant clones. The gene encoding the LOV-domain containing protein Dshi_1135 was identified by its colorless phenotype. The mutant phenotype was complemented by the expression of a Dshi_1135-strep fusion protein in trans. The recombinantly produced and chromatographically purified Dshi_1135 protein was able to undergo a blue light-induced photocycle mediated by bound FMN. Transcriptome analyses revealed an essential role for Dshi_1135 in the light-dependent expression of the photosynthetic gene cluster. Interactomic studies identified the repressor protein PpsR as an interaction partner of Dshi_1135. The physical contact between PpsR and the Dshi_1135 protein was verified in vivo using the bacterial adenylate cyclase-based two-hybrid system. In addition, the antirepressor function of the Dshi_1135 protein was demonstrated in vivo testing of a bchF-lacZ reporter gene fusion in a heterologous Escherichia coli-based host system. We therefore propose to rename the Dshi_1135 protein to LdaP (light-dependent antirepressor of PpsR). Using the bacterial two-hybrid system, it was also shown that cobalamin (B12) is essential for the interaction of the antirepressor PpaA with PpsR. A regulatory model for the photosynthetic gene cluster in D. shibae was derived, including the repressor PpsR, the light-dependent antirepressor LdaP and the B12-dependent antirepressor PpaA.

A systematic review of morphine equivalent conversions in plastic surgery: Current methods and future directions.

INTRODUCTION: Protocols surrounding opioid reduction have become commonplace in plastic surgery to improve peri-operative outcomes. Within such protocols, opioid requirement is a frequently analyzed outcome. Though often examined, there is no literature standard conversion for morphine milligram equivalents (MME) at present, leading to questionable external validity. We hypothesized significant heterogeneity in MME reporting would exist within plastic surgery literature. METHODS: Following the PRISMA guidelines, the authors conducted a systematic review of 16 journals. Clinical studies focused on opioid reduction within plastic surgery were identified. Primary outcomes included reporting of morphine equivalents (ME) delivery (IV/oral), operative ME, inpatient ME, outpatient ME, timeline, and method of calculation. RESULTS: Among the 101 studies analyzed, 73% reported opioid requirements in the form of ME. Among those that used ME, 3% reported IV ME, 41% reported oral, 32% reported both, and 25% gave no indication of either. Operative ME were reported in 19% of studies. Furthermore, 54% of studies reported inpatient ME whereas 32% of studies reported outpatient ME. Only 19% reported the number of days opioids were consumed postoperatively. Moreover, 27% of the studies reported the actual method of ME conversion, with 17 unique methods described. Only 8 studies (8%) reported using the Center for Disease Control and Prevention guidelines for ME conversion. CONCLUSION: There is significant variability among the reported ME conversion methodology within plastic surgery literature. Highlighting these discrepancies is an essential step in creating and implementing a single, standard method to mitigate opioid morbidity in plastic surgery and to optimize enhanced recovery protocols.

Halogenated Rocaglate Derivatives: Pan-antiviral Agents against Hepatitis E Virus and Emerging Viruses.

The synthesis of a library of halogenated rocaglate derivatives belonging to the flavagline class of natural products, of which silvestrol is the most prominent example, is reported. Their antiviral activity and cytotoxicity profile against a wide range of pathogenic viruses, including hepatitis E, Chikungunya, Rift Valley Fever virus and SARS-CoV-2, were determined. The incorporation of halogen substituents at positions 4', 6 and 8 was shown to have a significant effect on the antiviral activity of rocaglates, some of which even showed enhanced activity compared to CR-31-B and silvestrol.

Moving the needle: a narrative review of enhanced recovery protocols in breast reconstruction.

Background and Objective: After a relatively late introduction to the literature in 2015, enhanced recovery protocols for breast reconstruction have flourished into a wealth of reports. Many have since described unique methodologies making improved offerings with superior outcomes attainable. This is a particularly interesting procedure for the study of enhanced recovery as it encompasses two dissident approaches. Compared to implant-based reconstruction, autologous free-flap reconstruction has demonstrated superiority in a range of long-term metrics at the expense of historically increased peri-operative morbidity. This narrative review collates reports of recovery protocols for both approaches and examines methodologies surrounding the key pieces of a comprehensive pathway. Methods: All primary clinical reports specifically describing enhanced recovery protocols for implant-based and autologous breast reconstruction through 2022 were identified by systematic review of PubMed and Embase libraries. Twenty-five reports meeting criteria were identified, with ten additional reports included for narrative purpose. Included studies were examined for facets of innovation from the pre-hospital setting through outpatient follow-up. Notable findings were described in the context of a comprehensive framework with attention paid to clinical and basic scientific background. Considerations for implementation were additionally discussed. Key Content and Findings: Of 35 included studies, 29 regarded autologous reconstruction with majority focus on reduction of peri-operative opioid requirements and length of stay. Six regarded implant-based reconstruction with most discussing pathways towards ambulatory procedures. Eighty percent of included studies were published after the 2017 consensus guidelines with many described innovations to this baseline. Pathways included considerations for pre-hospital, pre-operative, intra-operative, inpatient, and outpatient settings. Implant-based studies demonstrated that safe ambulatory care is accessible. Autologous studies demonstrated a trend towards discharge before post-operative day three and peri-operative opioid requirements equivalent to those of implant-based reconstructions. Conclusions: Study of enhanced recovery after breast reconstruction has inspired paradigm shift and pushed limits previously not thought to be attainable. These protocols should encompass a longitudinal care pathway with optimization through patient-centered approaches and multidisciplinary collaboration. This framework should represent standard of care and will serve to expand availability of all methods of breast reconstruction.

Serine Protease Inhibitors Restrict Host Susceptibility to SARS-CoV-2 Infections.

The coronavirus disease 2019, COVID-19, is a complex disease with a wide range of symptoms from asymptomatic infections to severe acute respiratory syndrome with lethal outcome. Individual factors such as age, sex, and comorbidities increase the risk for severe infections, but other aspects, such as genetic variations, are also likely to affect the susceptibility to SARS-CoV-2 infection and disease severity. Here, we used a human 3D lung cell model based on primary cells derived from multiple donors to identity host factors that regulate SARS-CoV-2 infection. With a transcriptomics-based approach, we found that less susceptible donors show a higher expression level of serine protease inhibitors SERPINA1, SERPINE1, and SERPINE2, identifying variation in cellular serpin levels as restricting host factors for SARS-CoV-2 infection. We pinpoint their antiviral mechanism of action to inhibition of the cellular serine protease, TMPRSS2, thereby preventing cleavage of the viral spike protein and TMPRSS2-mediated entry into the target cells. By means of single-cell RNA sequencing, we further locate the expression of the individual serpins to basal, ciliated, club, and goblet cells. Our results add to the importance of genetic variations as determinants for SARS-CoV-2 susceptibility and suggest that genetic deficiencies of cellular serpins might represent risk factors for severe COVID-19. Our study further highlights TMPRSS2 as a promising target for antiviral intervention and opens the door for the usage of locally administered serpins as a treatment against COVID-19. IMPORTANCE Identification of host factors affecting individual SARS-CoV-2 susceptibility will provide a better understanding of the large variations in disease severity and will identify potential factors that can be used, or targeted, in antiviral drug development. With the use of an advanced lung cell model established from several human donors, we identified cellular protease inhibitors, serpins, as host factors that restrict SARS-CoV-2 infection. The antiviral mechanism was found to be mediated by the inhibition of a serine protease, TMPRSS2, which results in a blockage of viral entry into target cells. Potential treatments with these serpins would not only reduce the overall viral burden in the patients, but also block the infection at an early time point, reducing the risk for the hyperactive immune response common in patients with severe COVID-19.