Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions of OCA6, a tyrosine phosphatase gene, along with PMT1 or PMT2, two genes encoding ER membrane protein-O-mannosyltransferases involved in ER quality control, and SKI3, which encode for a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperone gene SCJ1 led to almost no secreted laccase activity. When we expressed SCJ1 from a low-copy plasmid, laccase secretion was restored. However, overexpression of SCJ1 had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.

Human retinal ganglion cell neurons generated by synchronous BMP inhibition and transcription factor mediated reprogramming.

In optic neuropathies, including glaucoma, retinal ganglion cells (RGCs) die. Cell transplantation and endogenous regeneration offer strategies for retinal repair, however, developmental programs required for this to succeed are incompletely understood. To address this, we explored cellular reprogramming with transcription factor (TF) regulators of RGC development which were integrated into human pluripotent stem cells (PSCs) as inducible gene cassettes. When the pioneer factor NEUROG2 was combined with RGC-expressed TFs (ATOH7, ISL1, and POU4F2) some conversion was observed and when pre-patterned by BMP inhibition, RGC-like induced neurons (RGC-iNs) were generated with high efficiency in just under a week. These exhibited transcriptional profiles that were reminiscent of RGCs and exhibited electrophysiological properties, including AMPA-mediated synaptic transmission. Additionally, we demonstrated that small molecule inhibitors of DLK/LZK and GCK-IV can block neuronal death in two pharmacological axon injury models. Combining developmental patterning with RGC-specific TFs thus provided valuable insight into strategies for cell replacement and neuroprotection.

Shift Work as a Cardiovascular Disease Risk Factor: A Narrative Review.

Shift work has emerged as a significant health concern in recent years, and research has revealed a link to circadian rhythm dysregulation and atherosclerosis, both of which can increase the risk of cardiovascular disease (CVD). Currently, there is a lack of updated reviews regarding the impact of shiftwork on CVD. Thus, the present narrative review aims to provide a comprehensive summary of the latest research on the relationship between shift work and CVD, identify potential gaps in the current knowledge, and highlight areas for future research. Database searches for peer-reviewed articles published between January 2013 to January 2023 on shift work associated CVD revealed many studies that found shift work is linked with increased prevalence of carotid artery plaque, increased arterial stiffness, and carotid artery intima-media thickness (IMT) all suggestive of a progression of atherosclerosis attributable to shift work. Hypertension, diabetes, and a sedentary lifestyle are known risks for CVD, and the results of the present study suggest that shift work should be added to that list. The elevation of inflammatory markers and DNA damage in shift workers may be linked to their increased progression of atherosclerosis and the positive association of shift work with coronary artery disease. There are minimal studies on mitigating approaches for shift work-related CVD, such as diet modification or exercise, emphasizing the need for further directed research in this area.

Sexual and Reproductive Health Among Men With Cystic Fibrosis.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Due to the distribution of the CFTR protein, CF presents with a heterogeneous phenotype. Men with CF may present with infertility due to congenital abnormalities of the vas deferens. In addition, they may experience testosterone deficiency. Today, they can father biological children with assisted reproductive technologies. We reviewed the current literature on the pathophysiology of these conditions, describe interventions that allow men with CF to conceive biological children, and provide recommendations for management of CF patients with reproductive health concerns.

Olfactory detection of viruses shapes brain immunity and behavior in zebrafish.

Olfactory sensory neurons (OSNs) are constantly exposed to pathogens, including viruses. However, serious brain infection via the olfactory route rarely occurs. When OSNs detect a virus, they coordinate local antiviral immune responses to stop virus progression to the brain. Despite effective immune control in the olfactory periphery, pathogen-triggered neuronal signals reach the CNS via the olfactory bulb (OB). We hypothesized that neuronal detection of a virus by OSNs initiates neuroimmune responses in the OB that prevent pathogen invasion. Using zebrafish ( Danio rerio ) as a model, we demonstrate viral-specific neuronal activation of OSNs projecting into the OB, indicating that OSNs are electrically activated by viruses. Further, behavioral changes are seen in both adult and larval zebrafish after viral exposure. By profiling the transcription of single cells in the OB after OSNs are exposed to virus, we found that both microglia and neurons enter a protective state. Microglia and macrophage populations in the OB respond within minutes of nasal viral delivery followed decreased expression of neuronal differentiation factors and enrichment of genes in the neuropeptide signaling pathway in neuronal clusters. Pituitary adenylate-cyclase-activating polypeptide ( pacap ), a known antimicrobial, was especially enriched in a neuronal cluster. We confirm that PACAP is antiviral in vitro and that PACAP expression increases in the OB 1 day post-viral treatment. Our work reveals how encounters with viruses in the olfactory periphery shape the vertebrate brain by inducing antimicrobial programs in neurons and by altering host behavior.

Human T cell generation is restored in CD3δ severe combined immunodeficiency through adenine base editing.

CD3δ SCID is a devastating inborn error of immunity caused by mutations in CD3D, encoding the invariant CD3δ chain of the CD3/TCR complex necessary for normal thymopoiesis. We demonstrate an adenine base editing (ABE) strategy to restore CD3δ in autologous hematopoietic stem and progenitor cells (HSPCs). Delivery of mRNA encoding a laboratory-evolved ABE and guide RNA into a CD3δ SCID patient's HSPCs resulted in a 71.2% ± 7.85% (n = 3) correction of the pathogenic mutation. Edited HSPCs differentiated in artificial thymic organoids produced mature T cells exhibiting diverse TCR repertoires and TCR-dependent functions. Edited human HSPCs transplanted into immunodeficient mice showed 88% reversion of the CD3D defect in human CD34+ cells isolated from mouse bone marrow after 16 weeks, indicating correction of long-term repopulating HSCs. These findings demonstrate the preclinical efficacy of ABE in HSPCs for the treatment of CD3δ SCID, providing a foundation for the development of a one-time treatment for CD3δ SCID patients.

The Impact of Nirmatrelvir-Ritonavir in Reducing Hospitalizations Among High-Risk Patients With SARS-CoV-2 During the Omicron Predominant Era.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has caused significant morbidity and mortality in high-risk populations. Several therapeutics have been developed to reduce the risk of complications related to COVID-19, hospitalizations, and death. In several studies, nirmatrelvir-ritonavir (NR) was reported to reduce the risk of hospitalizations and death. We aimed to evaluate the efficacy of NR in preventing hospitalizations and death during the Omicron predominant period. METHODS: We retrospectively evaluated patients from June 1, 2022, through September 24, 2022. There were a total of 25,939 documented COVID-19 cases. Using propensity matching, we matched 5754 patients treated with NR with untreated patients. RESULTS: Postmatching, the median age of the NR-treated group was 58 years (interquartile range, 43-70 years) and 42% were vaccinated. Postmatching composite outcome of the 30-day hospitalization and mortality in the NR-treated group were 0.9% (95% confidence interval [CI]: 0.7%-1.2%) versus 2.1% (95% CI: 1.8%-2.5%) in the matched control group, with a difference of -1.2 (-1.7, -0.8), P value <.01. The difference rates (NR vs. control) in 30-day all-cause hospitalizations and mortality were -1.2% (95% CI: -1.6% to -0.7%, P value <.01) and -0.1% (95% CI: -0.2% to 0.0%, P value = 0.29), respectively. We found similar finding across different age groups (≥65 vs. <65) and the vaccinated group. CONCLUSION: We report a significant benefit with the use of NR in reducing hospitalizations among various high-risk COVID-19 groups during the Omicron BA.5 predominant period.

Identification of newly translated thermo-sensitive proteins using pulse SILAC mass spectrometry and the GAL promoter system.

Some newly translated proteins are more susceptible to misfolding and aggregation upon heat shock in comparison to other proteins. To study these newly translated thermo-sensitive proteins on a proteomic scale, we present here a protocol that combines pulse-SILAC with biochemical fractionation for mass spectrometry analysis, followed by an orthogonal validation protocol for selected candidates using the GAL promoter system in Saccharomyces cerevisiae. This approach can be further developed to study other stresses and specific post-translational modifications or adapted to mammalian cells. For complete details on the use and execution of this protocol, please refer to Zhu et al. (2022).1.

Ecto-5'-nucleotidase (Nt5e/CD73)-mediated adenosine signaling attenuates TGFß-2 induced elastin and cellular contraction.

Arterial calcification due to deficiency of CD73 (ACDC) is a rare genetic disease caused by a loss-of-function mutation in the NT5E gene encoding the ecto-5'-nucleotidase (cluster of differentiation 73, CD73) enzyme. Patients with ACDC develop vessel arteriomegaly, tortuosity, and vascular calcification in their lower extremity arteries. Histological analysis shows that patients with ACDC vessels exhibit fragmented elastin fibers similar to that seen in aneurysmal-like pathologies. It is known that alterations in transforming growth factor ß (TGFß) pathway signaling contribute to this elastin phenotype in several connective tissue diseases, as TGFß regulates extracellular matrix (ECM) remodeling. Our study investigates whether CD73-derived adenosine modifies TGFß signaling in vascular smooth muscle cells (SMCs). We show that Nt5e-/- SMCs have elevated contractile markers and elastin gene expression compared with Nt5e+/+ SMCs. Ecto-5'-nucleotidase (Nt5e)-deficient SMCs exhibit increased TGFß-2 and activation of small mothers against decapentaplegic (SMAD) signaling, elevated elastin transcript and protein, and potentiate SMC contraction. These effects were diminished when the A2b adenosine receptor was activated. Our results identify a novel link between adenosine and TGFß signaling, where adenosine signaling via the A2b adenosine receptor attenuates TGFß signaling to regulate SMC homeostasis. We discuss how disruption in adenosine signaling is implicated in ACDC vessel tortuosity and could potentially contribute to other aneurysmal pathogenesis.

Lentiviral gene therapy for X-linked chronic granulomatous disease recapitulates endogenous CYBB regulation and expression.

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene, resulting in the inability of phagocytic cells to eliminate infections. To design a lentiviral vector (LV) capable of recapitulating the endogenous regulation and expression of CYBB, a bioinformatics-guided approach was used to elucidate the cognate enhancer elements regulating the native CYBB gene. Using this approach, we analyzed a 600-kilobase topologically associated domain of the CYBB gene and identified endogenous enhancer elements to supplement the CYBB promoter to develop MyeloVec, a physiologically regulated LV for the treatment of X-CGD. When compared with an LV currently in clinical trials for X-CGD, MyeloVec showed improved expression, superior gene transfer to hematopoietic stem and progenitor cells (HSPCs), corrected an X-CGD mouse model leading to complete protection against Burkholderia cepacia infection, and restored healthy donor levels of antimicrobial oxidase activity in neutrophils derived from HSPCs from patients with X-CGD. Our findings validate the bioinformatics-guided design approach and have yielded a novel LV with clinical promise for the treatment of X-CGD.

A Tet-Inducible CRISPR Platform for High-Fidelity Editing of Human Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) offer an exciting resource for probing human biology; however, gene-editing efficiency remains relatively low in many cell types, including stem cells. Gene-editing using the CRISPR-Cas9 system offers an attractive solution that improves upon previous gene-editing approaches; however, like other technologies, off-target mutagenesis remains a concern. High-fidelity Cas9 variants greatly reduce off-target mutagenesis and offer a solution to this problem. To evaluate their utility as part of a cell-based gene-editing platform, human PSC lines were generated with a high-fidelity (HF) tetracycline-inducible engineered Streptococcus pyogenes SpCas9 (HF-iCas9) integrated into the AAVS1 safe harbor locus. By engineering cells with controllable expression of Cas9, we eliminated the need to include a large Cas9-expressing plasmid during cell transfection. Delivery of genetic cargo was further optimized by packaging DNA targeting guide RNAs (gRNAs) and donor fragments into a single plasmid backbone. The potential of homology-directed repair (HDR) based gene knock-in at the CLYBL safe harbor site and endogenous SOX2 and SIX6 genes were demonstrated. Moreover, we used non-homologous end-joining (NHEJ) for gene knockout of disease-relevant alleles. These high-fidelity CRISPR tools and the resulting HF-iCas9 cell lines will facilitate the production of cell-type reporters and mutants across different genetic backgrounds.

Circular RNAs in organ injury: recent development.

Circular ribonucleic acids (circRNAs) are a class of long non-coding RNA that were once regarded as non-functional transcription byproducts. However, recent studies suggested that circRNAs may exhibit important regulatory roles in many critical biological pathways and disease pathologies. These studies have identified significantly differential expression profiles of circRNAs upon changes in physiological and pathological conditions of eukaryotic cells. Importantly, a substantial number of studies have suggested that circRNAs may play critical roles in organ injuries. This review aims to provide a summary of recent studies on circRNAs in organ injuries with respect to (1) changes in circRNAs expression patterns, (2) main mechanism axi(e)s, (3) therapeutic implications and (4) future study prospective. With the increasing attention to this research area and the advancement in high-throughput nucleic acid sequencing techniques, our knowledge of circRNAs may bring fruitful outcomes from basic and clinical research.

Melamine-graphene epoxy nanocomposite based die attach films for advanced 3D semiconductor packaging applications.

With the ultra-fast development of personal portable electronic devices, it is important to explore new die attach film (DAF) materials in the limited mounting area and height in order to meet the requirements of a high packaging density and a high operating speed. Graphene-based epoxy nanocomposites are becoming one of the most promising candidates for the next generation of DAFs combining the ultra-high thermal conductivity of graphene, and ultra-strong adhesion of epoxy polymers. However, poor dispersion and weak interfacial connections, due to the overly smooth surface of graphene nanosheets, are still pressing issues that limit their industrial applications. Additionally, pristine graphene nanosheets have only a small effect on improving the glass transition temperature (Tg) of epoxy composites to meet the requirements of DAFs. In this work, melamine-functionalized graphene is synthesized by using a nondestructive ball milling process, which results in greater dispersion and enhancement of the interfacial connections between graphene and epoxy resins demonstrated by both experimental and simulation results. In particular, the aromatic triazine rings of melamine increase Tg in the cured resin, thus improving the thermal stability of DAFs. The melamine-graphene (M-G) epoxy nanocomposites synthesized have a high Tg of 172 °C and an out-of-plane thermal conductivity of 1.08 W m-1 K-1 at 10 wt% loading. This is 6.4 multiples higher than that of neat epoxy. Moreover, M-G epoxy nanocomposites exhibit superb thermal stability, an effective low coefficient of thermal expansion (CTE), low moisture adsorption, and a useful high electrical resistivity. In the DAF performance test, involving experimentation and modeling, the samples present a better cooling capability and heat dissipation. This supports the idea that our findings have potential to be applied in the next generation of DAFs for high-power and high-density 3D packaging.

Pulse labeling reveals the tail end of protein folding by proteome profiling.

Accurate and efficient folding of nascent protein sequences into their native states requires support from the protein homeostasis network. Herein we probe which newly translated proteins are thermo-sensitive, making them susceptible to misfolding and aggregation under heat stress using pulse-SILAC mass spectrometry. We find a distinct group of proteins that is highly sensitive to this perturbation when newly synthesized but not once matured. These proteins are abundant and highly structured. Notably, they display a tendency to form ß sheet secondary structures, have more complex folding topology, and are enriched for chaperone-binding motifs, suggesting a higher demand for chaperone-assisted folding. These polypeptides are also more often components of stable protein complexes in comparison with other proteins. Combining these findings suggests the existence of a specific subset of proteins in the cell that is particularly vulnerable to misfolding and aggregation following synthesis before reaching the native state.

Young age is associated with decreased recurrence for renal cell carcinoma.

INTRODUCTION: We aimed to examine stage-specific oncologic outcomes for young versus conventional-age patients with localized disease in a modern cohort. MATERIALS AND METHODS: The Surveillance, Epidemiology and End Results database was queried for patients with T1-T2N0M0 kidney cancer from 1975-2016, including clear cell, papillary, and chromophobe renal cell carcinoma. Patients were stratified into ≤ 40 years-old or > 40 years-old cohorts and underwent definitive treatment via percutaneous ablation, partial nephrectomy, or radical nephrectomy. Primary outcome was cancer-specific survival. Cox regression and Kaplan-Meier analysis were performed. RESULTS: A total of 44,673 patients were identified with 41,812 patients in the conventional-age and 2,861 patients in the young cohort with mean ages of 62.1 and 34.7 years old, respectively. The young cohort had a higher proportion of T1a disease compared to the conventional-age cohort (65.2% vs. 58.6%) and a lower proportion of the cT1b (24.4% vs. 29.3%), cT2a (6.8% vs. 8.4%), and cT2b (3.6% vs. 3.7%) disease. Chromophobe histology was more prevalent in the younger population (10.5% vs. 6.6%). Nuclear grade 3 or 4 were more prominent in the conventional-age population (24.8% vs. 19.1%). Cancer-specific death was significantly higher in the conventional-age cohort (2.4% vs. 0.7%). Cox regression analysis demonstrated patients > 40 years old, increasing stage, and higher grade were at independently increased risk of cancer-specific death. Kaplan-Meier analysis showed significantly improved 5-year cancer-specific survival for the young versus conventional-age cohorts when sub-stratified by stage. CONCLUSION: When stratified by stage, young patients with localized kidney cancer experience improved cancer-specific survival.

Increasing rate of pathologic upgrading in low risk prostate cancer patients in the active surveillance era.

INTRODUCTION: Management of prostate cancer has seen an increasing predilection for active surveillance in low risk (LR) patients. We aimed to evaluate the rate of pathologic upgrading in patients with very low (VLR) or LR prostate cancer after prostatectomy. MATERIALS AND METHODS: The National Cancer Database (NCDB) and the Surveillance, Epidemiology, and End Results (SEER) Database were queried for patients diagnosed with Gleason 6 prostate cancer and prostate specific antigen (PSA) < 10 ng/mL from 2010 to 2016. All patients underwent 12-core biopsy and a subsequent prostatectomy for final pathologic staging. Our primary outcome was rate of pathologic upgrading over the study period. RESULTS: A total of 35,332 patients from the NCDB and 7,186 patients from the SEER database were collected. Patient population had an average age of about 59 years old and was over 80% white. Mean pre-biopsy PSA was higher for the upgraded cohorts in the NCDB and SEER populations (5.3 versus 4.9 and 5.5 versus 5.1 respectively, p < 0.001). Upgraded cohorts were more likely to have a higher percentage of positive cores at biopsy (p < 0.001). Multivariable analysis demonstrated that increasing age, increasing PSA and year of diagnosis were all predictors of upgrading (p < 0.05) in both databases. African American race was significantly associated with upgrading in the NCDB database only (p = 0.001). Over the studied time period, the rate of upgrading at prostatectomy increased from 41.2% to 56.7% in the NCDB population and from 41.9% to 45.4% in the SEER population. CONCLUSIONS: The rate of pathologic upgrading of VLR and LR prostate cancer at prostatectomy has been increasing in recent years. Increasing age, pre-biopsy PSA and an increasing percentage of positive cores at biopsy are predictors of this outcome. This may relate to improved patient selection for active surveillance and definitive treatment.

The Clinical Utility of Two High-Throughput 16S rRNA Gene Sequencing Workflows for Taxonomic Assignment of Unidentifiable Bacterial Pathogens in Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Bacterial pathogens that cannot be identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) are occasionally encountered in clinical laboratories. The 16S rRNA gene is often used for sequence-based analysis to identify these bacterial species. Nevertheless, traditional Sanger sequencing is laborious, time-consuming, and low throughput. Here, we compared two commercially available 16S rRNA gene sequencing tests that are based on Illumina and Nanopore sequencing technologies, respectively, in their ability to identify the species of 172 clinical isolates that failed to be identified by MALDI-TOF MS. Sequencing data were analyzed by the respective built-in programs (MiSeq Reporter software of Illumina and Epi2me of Nanopore) and BLAST+ (v2.11.0). Their agreement with Sanger sequencing on species-level identification was determined. Discrepancies were resolved by whole-genome sequencing. The diagnostic accuracy of each workflow was determined using the composite sequencing result as the reference standard. Despite the high base-calling accuracy of Illumina sequencing, we demonstrated that the Nanopore workflow had a higher taxonomic resolution at the species level. Using built-in analysis algorithms, the concordance of Sanger 16S with the Illumina and Nanopore workflows was 33.14% and 87.79%, respectively. The agreement was 65.70% and 83.14%, respectively, when BLAST+ was used for analysis. Compared with the reference standard, the diagnostic accuracy of Nanopore 16S was 96.36%, which was identical to that of Sanger 16S and better than that of Illumina 16S (69.07%). The turnaround time of the Illumina workflow and the Nanopore workflow was 78 h and 8.25 h, respectively. The per-sample cost of the Illumina and Nanopore workflows was US$28.5 and US$17.7, respectively.

Engineering Cancer Antigen-Specific T Cells to Overcome the Immunosuppressive Effects of TGF-ß.

Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-ß, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-ß. Truncating the intracellular signaling domain from TGF-ß receptor (TGFßR) II produces a dominant-negative receptor (dnTGFßRII) that dimerizes with endogenous TGFßRI to form a receptor that can bind TGF-ß but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02-restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157-165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254-262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-ß inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFßRII (e.g., GSK3845097). TGF-ß isoforms and a panel of TGF-ß-associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-ß-positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non-small cell lung cancer setting. Coexpression of dnTGFßRII may therefore improve the efficacy of TCR-transduced T cells.