A 5' UTR Language Model for Decoding Untranslated Regions of mRNA and Function Predictions.

The 5' UTR, a regulatory region at the beginning of an mRNA molecule, plays a crucial role in regulating the translation process and impacts the protein expression level. Language models have showcased their effectiveness in decoding the functions of protein and genome sequences. Here, we introduced a language model for 5' UTR, which we refer to as the UTR-LM. The UTR-LM is pre-trained on endogenous 5' UTRs from multiple species and is further augmented with supervised information including secondary structure and minimum free energy. We fine-tuned the UTR-LM in a variety of downstream tasks. The model outperformed the best known benchmark by up to 5% for predicting the Mean Ribosome Loading, and by up to 8% for predicting the Translation Efficiency and the mRNA Expression Level. The model also applies to identifying unannotated Internal Ribosome Entry Sites within the untranslated region and improves the AUPR from 0.37 to 0.52 compared to the best baseline. Further, we designed a library of 211 novel 5' UTRs with high predicted values of translation efficiency and evaluated them via a wet-lab assay. Experiment results confirmed that our top designs achieved a 32.5% increase in protein production level relative to well-established 5' UTR optimized for therapeutics.

Three-Dimensional Laparoscopic Nephrectomy for Benign Nonfunctioning Kidneys: A Single-Center Initial Experience.

BACKGROUND: There are several types of benign renal diseases, such as urological stones, ureteropelvic junction obstruction, renal vascular disease, and inflammation, which are responsible for nonfunctioning kidneys. Laparoscopic nephrectomy (LN) is the gold standard for treating nonfunctioning kidneys with complications. This study presents the results of our initial experiences with 3D laparoscopic nephrectomy (3D-LN) for benign, nonfunctioning kidneys. METHODS: From July 2021 to July 2023, 40 consecutive patients who underwent 3D transperitoneal laparoscopic nephrectomy were retrospectively evaluated at the Department of Urology and Department of General Surgery, Hue Central Hospital, Hue, Vietnam. Patient demographics, intraoperative and early postoperative results, postoperative recovery, complications, and three-month follow-up results were recorded. RESULTS: The mean age was 58.35 ± 14.9 years. There were 13 (32.5%) male and 27 (67.5%) female patients. Flank pain was the main reason for hospitalization in 33 cases (82.5%); the common cause of a nonfunctioning kidney was urological stones (62.5%). Twenty-three out of 40 patients underwent a left nephrectomy. The average operative time was 92.57 ± 28.69 minutes. A statistically significant difference in surgery time was found between the group with no adhesion and the group with mild adhesion, as well as between the first 19 patients and the last 18 patients (p <0.05). The mean blood loss was 51.62 ± 24.35 ml. Three cases were converted to open surgery due to severe adhesions. The postoperative complications rate was 8.1%. The average length of the postoperative hospital stay was 7.89 ± 3.59 days. CONCLUSIONS: Three-dimensional laparoscopic nephrectomy is a safe and effective method that increases depth perception and spatial orientation for surgeons and can compensate for the remaining shortcomings of traditional 2D systems.

Efficient and multiplexed somatic genome editing with Cas12a mice.

Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations in areas ranging from neuroscience to cancer biology and beyond. However, existing models are limited in their ability to create multiple targeted edits. Thus, our understanding of the complex genetic interactions that underlie development, homeostasis, and disease remains incomplete. Cas12a is an RNA-guided endonuclease with unique attributes that enable simple targeting of multiple genes with crRNA arrays containing tandem guides. To accelerate and expand the generation of complex genotypes in somatic cells, we generated transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a). In these mice, enAsCas12a-mediated somatic genome editing robustly generated compound genotypes, as exemplified by the initiation of diverse cancer types driven by homozygous inactivation of trios of tumor suppressor genes. We further integrated these modular crRNA arrays with clonal barcoding to quantify the size and number of tumors with each array, as well as the efficiency of each crRNA. These Cas12a alleles will enable the rapid generation of disease models and broadly facilitate the high-throughput investigation of coincident genomic alterations in somatic cells in vivo .

APOE loss-of-function variants: Compatible with longevity and associated with resistance to Alzheimer's disease pathology.

The ε4 allele of apolipoprotein E (APOE) is the strongest genetic risk factor for sporadic Alzheimer's disease (AD). Knockdown of ε4 may provide a therapeutic strategy for AD, but the effect of APOE loss of function (LoF) on AD pathogenesis is unknown. We searched for APOE LoF variants in a large cohort of controls and patients with AD and identified seven heterozygote carriers of APOE LoF variants. Five carriers were controls (aged 71-90 years), one carrier was affected by progressive supranuclear palsy, and one carrier was affected by AD with an unremarkable age at onset of 75 years. Two APOE ε3/ε4 controls carried a stop-gain affecting ε4: one was cognitively normal at 90 years and had no neuritic plaques at autopsy; the other was cognitively healthy at 79 years, and lumbar puncture at 76 years showed normal levels of amyloid. These results suggest that ε4 drives AD risk through the gain of abnormal function and support ε4 knockdown as a viable therapeutic option.

The Outcome of the Miniaturized Percutaneous Nephrolithotomy in Lateral Position with Guidance of Non-Rotational C-Arm.

Objective: Evaluation of the outcome of the miniaturized percutaneous nephrolithotomy in the treatment of nephrolithiasis with a lateral position based on the principle of a right triangle under the guidance of a C-Arm without contrast fluid. Materials and Methods: Sixty-nine cases of Mini-PCNL with the assistance of a vacuum-assisted sheath in the lateral position were performed at Central Hospital from March 2021 to August 2022. Percutaneous renal access was under the guidance of a non-rotational C-arm without contrast medium, and we determined the puncture location and depth of the needle based on the principle of right triangles. Results: The median age was 51.6 ± 12.5 years, and males accounted for 68.1% of the cases. 60.9% of all patients had normal weight. The median stone surface area was 361.1mm2, and 59.4% of all cases were graded as 2 regarding Guy's stone score. The successful renal access rate was 100%. The tunnel access from the middle and lower calyx accounted for 94.2%. The median access duration, fluoroscopy duration, and hospital length of stay were 271.7 seconds, 14.79 seconds, and 6.3 days, respectively. The complete stone clearance rate was 78.3%. Bleeding complications occurred in 2 patients without mortality. Three patients required an additional procedure. Conclusion: The puncture technique into the renal calyxes based on the principle of the right triangle under the guidance of a non-rotational C-Arm without contrast medium in PCNL is a fast, exact, and safe technique.

Integrative analysis of functional genomic screening and clinical data identifies a protective role for spironolactone in severe COVID-19.

We demonstrate that integrative analysis of CRISPR screening datasets enables network-based prioritization of prescription drugs modulating viral entry in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by developing a network-based approach called Rapid proXimity Guidance for Repurposing Investigational Drugs (RxGRID). We use our results to guide a propensity-score-matched, retrospective cohort study of 64,349 COVID-19 patients, showing that a top candidate drug, spironolactone, is associated with improved clinical prognosis, measured by intensive care unit (ICU) admission and mechanical ventilation rates. Finally, we show that spironolactone exerts a dose-dependent inhibitory effect on viral entry in human lung epithelial cells. Our RxGRID method presents a computational framework, implemented as an open-source software package, enabling genomics researchers to identify drugs likely to modulate a molecular phenotype of interest based on high-throughput screening data. Our results, derived from this method and supported by experimental and clinical analysis, add additional supporting evidence for a potential protective role of the potassium-sparing diuretic spironolactone in severe COVID-19.

APOE loss-of-function variants: Compatible with longevity and associated with resistance to Alzheimer's Disease pathology.

The ε4 allele of apolipoprotein E (APOE) is the strongest genetic risk factor for sporadic Alzheimer's Disease (AD). Knockdown of this allele may provide a therapeutic strategy for AD, but the effect of APOE loss-of-function (LoF) on AD pathogenesis is unknown. We searched for APOE LoF variants in a large cohort of older controls and patients with AD and identified six heterozygote carriers of APOE LoF variants. Five carriers were controls (ages 71-90) and one was an AD case with an unremarkable age-at-onset between 75-79. Two APOE ε3/ε4 controls (Subjects 1 and 2) carried a stop-gain affecting the ε4 allele. Subject 1 was cognitively normal at 90+ and had no neuritic plaques at autopsy. Subject 2 was cognitively healthy within the age range 75-79 and underwent lumbar puncture at between ages 75-79 with normal levels of amyloid. The results provide the strongest human genetics evidence yet available suggesting that ε4 drives AD risk through a gain of abnormal function and support knockdown of APOE ε4 or its protein product as a viable therapeutic option.

High Prevalence of Post-Traumatic Stress Disorder and Psychological Distress Among Healthcare Workers in COVID-19 Field Hospitals: A Cross-Sectional Study from Vietnam.

Objective: To evaluate the prevalence of post-traumatic stress disorder (PTSD) and other psychological disturbances in the Vietnamese healthcare workers (HCWs) at COVID-19 field hospitals. Methods: A cross-sectional study was conducted using the Impact of Event Scale-Revised (IES-R) to measure PTSD and the Depression Anxiety Stress scale (DASS) to measure other psychological disturbances. The anxiety about COVID-19 was evaluated by the fear of COVID-19 (FOC) scale. A self-developed questionnaire was used to assess work conditions and HCW's major concerns and preparedness. Ordinal logistic regression was used to identify factors associated with the severity of PTSD. A structural modeling equation (SEM) model was fitted to examine the correlation between PTSD and other psychological disturbances. Results: A total of 542 HCWs participated in this study. The prevalence of PTSD was 21.2%, most cases were mild. In the ordinal logistic regression analysis, a history of mental illness, poor preparedness, working in a condition with poor resources, a greater number of concerns, and greater fear of COVID-19 were independently associated with higher severity of PTSD. The prevalence of depression, anxiety, and stress was 46.8%, 38.3%, and 60.2, respectively. In the SEM model, PTSD and psychological disturbances had a strong correlation (standardized covariance 0.86). Conclusion: The prevalence of PTSD and other psychological disturbances was alarmingly high among HCWs who worked at COVID-19 field hospitals. The reported associated factors can be useful for policymakers and health authorities in the preparation for future pandemics.

Evaluation of polyhydroxyalkanoate (PHA) synthesis by Pichia sp. TSLS24 yeast isolated in Vietnam.

Following the rising concern on environmental issues caused by conventional fossil-based plastics and depleting crude oil resources, polyhydroxyalkanoates (PHAs) are of great interest by scientists and biodegradable polymer market due to their outstanding properties which include high biodegradability in various conditions and processing flexibility. Many polyhydroxyalkanoate-synthesizing microorganisms, including normal and halophilic bacteria, as well as algae, have been investigated for their performance in polyhydroxyalkanoate production. However, to the best of our knowledge, there is still limited studies on PHAs-producing marine yeast. In the present study, a halophilic yeast strain isolated from Spratly Island in Vietnam were investigated for its potential in polyhydroxyalkanoate biosynthesis by growing the yeast in Zobell marine agar medium (ZMA) containing Nile red dye. The strain was identified by 26S rDNA analysis as Pichia kudriavzevii TSLS24 and registered at Genbank database under code OL757724. The amount of polyhydroxyalkanoates synthesized was quantified by measuring the intracellular materials (predicted as poly(3-hydroxybutyrate) -PHB) by gravimetric method and subsequently confirmed by Fourier transform infrared (FTIR) spectroscopic and nuclear magnetic resonance (NMR) spectroscopic analyses. Under optimal growth conditions of 35 °C and pH 7 with supplementation of glucose and yeast extract at 20 and 10 gL-1, the isolated strain achieved poly(3-hydroxybutyrate) content and concentration of 43.4% and 1.8 gL-1 after 7 days of cultivation. The poly(3-hydroxybutyrate) produced demonstrated excellent biodegradability with degradation rate of 28% after 28 days of incubation in sea water.

Gene set proximity analysis: expanding gene set enrichment analysis through learned geometric embeddings, with drug-repurposing applications in COVID-19.

MOTIVATION: Gene set analysis methods rely on knowledge-based representations of genetic interactions in the form of both gene set collections and protein-protein interaction (PPI) networks. However, explicit representations of genetic interactions often fail to capture complex interdependencies among genes, limiting the analytic power of such methods. RESULTS: We propose an extension of gene set enrichment analysis to a latent embedding space reflecting PPI network topology, called gene set proximity analysis (GSPA). Compared with existing methods, GSPA provides improved ability to identify disease-associated pathways in disease-matched gene expression datasets, while improving reproducibility of enrichment statistics for similar gene sets. GSPA is statistically straightforward, reducing to a version of traditional gene set enrichment analysis through a single user-defined parameter. We apply our method to identify novel drug associations with SARS-CoV-2 viral entry. Finally, we validate our drug association predictions through retrospective clinical analysis of claims data from 8 million patients, supporting a role for gabapentin as a risk factor and metformin as a protective factor for severe COVID-19. AVAILABILITY AND IMPLEMENTATION: GSPA is available for download as a command-line Python package at https://github.com/henrycousins/gspa. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Long sequence insertion via CRISPR/Cas gene-editing with transposase, recombinase, and integrase.

CRISPR/Cas-based gene-editing technologies have emerged as one of the most transformative tools in genome science over the past decade, providing unprecedented possibilities for both fundamental and translational research. Following the initial wave of innovations for gene knock-out, epigenetic/RNA modulation, and nickase-mediated base-editing, recent efforts have pivoted towards long-sequence gene editing- specifically, the insertion of large fragments (>1 kb) into the endogenous genome. In this review, we survey the development of these CRISPR/Cas-based sequence insertion methodologies in conjunction with the emergence of novel families of editing enzymes, such as transposases, single-stranded DNA-annealing proteins, recombinases, and integrases. Despite facing a number of challenges, this field continues to evolve rapidly and holds the potential to catalyze a new wave of revolutionary biomedical applications.

Single-cell transcriptome analysis of regenerating RGCs reveals potent glaucoma neural repair genes.

Axon regeneration holds great promise for neural repair of CNS axonopathies, including glaucoma. Pten deletion in retinal ganglion cells (RGCs) promotes potent optic nerve regeneration, but only a small population of Pten-null RGCs are actually regenerating RGCs (regRGCs); most surviving RGCs (surRGCs) remain non-regenerative. Here, we developed a strategy to specifically label and purify regRGCs and surRGCs, respectively, from the same Pten-deletion mice after optic nerve crush, in which they differ only in their regeneration capability. Smart-Seq2 single-cell transcriptome analysis revealed novel regeneration-associated genes that significantly promote axon regeneration. The most potent of these, Anxa2, acts synergistically with its ligand tPA in Pten-deletion-induced axon regeneration. Anxa2, its downstream effector ILK, and Mpp1 dramatically protect RGC somata and axons and preserve visual function in a clinically relevant model of glaucoma, demonstrating the exciting potential of this innovative strategy to identify novel effective neural repair candidates.

Machine-learning-optimized Cas12a barcoding enables the recovery of single-cell lineages and transcriptional profiles.

The development of CRISPR-based barcoding methods creates an exciting opportunity to understand cellular phylogenies. We present a compact, tunable, high-capacity Cas12a barcoding system called dual acting inverted site array (DAISY). We combined high-throughput screening and machine learning to predict and optimize the 60-bp DAISY barcode sequences. After optimization, top-performing barcodes had ∼10-fold increased capacity relative to the best random-screened designs and performed reliably across diverse cell types. DAISY barcode arrays generated ∼12 bits of entropy and ∼66,000 unique barcodes. Thus, DAISY barcodes-at a fraction of the size of Cas9 barcodes-achieved high-capacity barcoding. We coupled DAISY barcoding with single-cell RNA-seq to recover lineages and gene expression profiles from ∼47,000 human melanoma cells. A single DAISY barcode recovered up to ∼700 lineages from one parental cell. This analysis revealed heritable single-cell gene expression and potential epigenetic modulation of memory gene transcription. Overall, Cas12a DAISY barcoding is an efficient tool for investigating cell-state dynamics.

dCas9-based gene editing for cleavage-free genomic knock-in of long sequences.

Gene editing is a powerful tool for genome and cell engineering. Exemplified by CRISPR-Cas, gene editing could cause DNA damage and trigger DNA repair processes that are often error-prone. Such unwanted mutations and safety concerns can be exacerbated when altering long sequences. Here we couple microbial single-strand annealing proteins (SSAPs) with catalytically inactive dCas9 for gene editing. This cleavage-free gene editor, dCas9-SSAP, promotes the knock-in of long sequences in mammalian cells. The dCas9-SSAP editor has low on-target errors and minimal off-target effects, showing higher accuracy than canonical Cas9 methods. It is effective for inserting kilobase-scale sequences, with an efficiency of up to approximately 20% and robust performance across donor designs and cell types, including human stem cells. We show that dCas9-SSAP is less sensitive to inhibition of DNA repair enzymes than Cas9 references. We further performed truncation and aptamer engineering to minimize its size to fit into a single adeno-associated-virus vector for future application. Together, this tool opens opportunities towards safer long-sequence genome engineering.

Conventional type I dendritic cells maintain a reservoir of proliferative tumor-antigen specific TCF-1+ CD8+ T cells in tumor-draining lymph nodes.

In tumors, a subset of CD8+ T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1+ CD8+ T cells revealed that while intratumoral TCF-1+ CD8+ T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1+ CD8+ T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1+ CD8+ T cell subsets developed over time-a proliferative SlamF6+ subset and a non-cycling SlamF6- subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6+ TCF-1+ CD8+ T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6+ T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1+ CD8+ T cells and their decrease contributes to failed anti-tumor immunity.

Deciphering pathogenicity of variants of uncertain significance with CRISPR-edited iPSCs.

Genetic variants play an important role in conferring risk for cardiovascular diseases (CVDs). With the rapid development of next-generation sequencing (NGS), thousands of genetic variants associated with CVDs have been identified by genome-wide association studies (GWAS), but the function of more than 40% of genetic variants is still unknown. This gap of knowledge is a barrier to the clinical application of the genetic information. However, determining the pathogenicity of a variant of uncertain significance (VUS) is challenging due to the lack of suitable model systems and accessible technologies. By combining clustered regularly interspaced short palindromic repeats (CRISPR) and human induced pluripotent stem cells (iPSCs), unprecedented advances are now possible in determining the pathogenicity of VUS in CVDs. Here, we summarize recent progress and new strategies in deciphering pathogenic variants for CVDs using CRISPR-edited human iPSCs.

Advanced materials for immobilization of purple phototrophic bacteria in bioremediation of oil-polluted wastewater.

Oil pollution which results from industrial activities, especially oil and gas industry, has become a serious issue. Cinder beats (CB), coconut fiber (CF) and polyurethane foam (PUF) are promising immobilization carriers for crude oil biodegradation because they are inexpensive, nontoxic, and non-polluting. The present investigation was aimed to evaluate this advanced technology and compare the efficiency of these immobilization carriers on supporting purple phototrophic bacterial (PPB) strains in hydrocarbon biodegradation of crude oil contaminated seawater. The surface of these biocarriers was supplemented with crude oil polluted seawater and immobilized by PPB strains, Rhodopseudomonas sp. DD4, DQ41 and FO2. Through scanning electron microscopy (SEM), the bacterial cells were shown to colonize and attach strongly to these biocarriers. The bacteria-driven carrier systems degraded over 84.2% supplemented single polycyclic aromatic hydrocarbons (PAHs). The aliphatic and aromatic components in crude oil that treated with carrier-immobilized consortia were degraded remarkably after 14 day-incubation. Among the three biocarriers, removal of the crude oil by CF-bacteria system was the highest (nearly 100%), followed by PUF-bacteria (89.5%) and CB-bacteria (86.3%) with the initial crude oil concentration was 20 g/L. Efficiency of crude oil removal by CB-bacteria and PUF-bacteria were 86.3 and 89.5%, respectively. Till now, the studies on crude oil degradation by mixture species biofilms formed by PPB on different carriers are limited. The present study showed that the biocarriers of an oil-degrading consortium could be made up of waste materials that are cheap and eco-friendly as well as augment the biodegradation of oil-contaminated seawater.

A Functional Taxonomy of Tumor Suppression in Oncogenic KRAS-Driven Lung Cancer.

Cancer genotyping has identified a large number of putative tumor suppressor genes. Carcinogenesis is a multistep process, but the importance and specific roles of many of these genes during tumor initiation, growth, and progression remain unknown. Here we use a multiplexed mouse model of oncogenic KRAS-driven lung cancer to quantify the impact of 48 known and putative tumor suppressor genes on diverse aspects of carcinogenesis at an unprecedented scale and resolution. We uncover many previously understudied functional tumor suppressors that constrain cancer in vivo. Inactivation of some genes substantially increased growth, whereas the inactivation of others increases tumor initiation and/or the emergence of exceptionally large tumors. These functional in vivo analyses revealed an unexpectedly complex landscape of tumor suppression that has implications for understanding cancer evolution, interpreting clinical cancer genome sequencing data, and directing approaches to limit tumor initiation and progression. SIGNIFICANCE: Our high-throughput and high-resolution analysis of tumor suppression uncovered novel genetic determinants of oncogenic KRAS-driven lung cancer initiation, overall growth, and exceptional growth. This taxonomy is consistent with changing constraints during the life history of cancer and highlights the value of quantitative in vivo genetic analyses in autochthonous cancer models.This article is highlighted in the In This Issue feature, p. 1601.