Accelerating ionizable lipid discovery for mRNA delivery using machine learning and combinatorial chemistry.

To unlock the full promise of messenger (mRNA) therapies, expanding the toolkit of lipid nanoparticles is paramount. However, a pivotal component of lipid nanoparticle development that remains a bottleneck is identifying new ionizable lipids. Here we describe an accelerated approach to discovering effective ionizable lipids for mRNA delivery that combines machine learning with advanced combinatorial chemistry tools. Starting from a simple four-component reaction platform, we create a chemically diverse library of 584 ionizable lipids. We screen the mRNA transfection potencies of lipid nanoparticles containing those lipids and use the data as a foundational dataset for training various machine learning models. We choose the best-performing model to probe an expansive virtual library of 40,000 lipids, synthesizing and experimentally evaluating the top 16 lipids flagged. We identify lipid 119-23, which outperforms established benchmark lipids in transfecting muscle and immune cells in several tissues. This approach facilitates the creation and evaluation of versatile ionizable lipid libraries, advancing the formulation of lipid nanoparticles for precise mRNA delivery.

Combinatorial development of nebulized mRNA delivery formulations for the lungs.

Inhaled delivery of mRNA has the potential to treat a wide variety of diseases. However, nebulized mRNA lipid nanoparticles (LNPs) face several unique challenges including stability during nebulization and penetration through both cellular and extracellular barriers. Here we develop a combinatorial approach addressing these barriers. First, we observe that LNP formulations can be stabilized to resist nebulization-induced aggregation by altering the nebulization buffer to increase the LNP charge during nebulization, and by the addition of a branched polymeric excipient. Next, we synthesize a combinatorial library of ionizable, degradable lipids using reductive amination, and evaluate their delivery potential using fully differentiated air-liquid interface cultured primary lung epithelial cells. The final combination of ionizable lipid, charge-stabilized formulation and stability-enhancing excipient yields a significant improvement in lung mRNA delivery over current state-of-the-art LNPs and polymeric nanoparticles.

Normal central foveal thickness in a thousand eyes of healthy patients in sub Saharan Africa using fourier domain optical coherence tomography.

Background: Optical coherence tomography provides high resolution in vivo images of the retina which are essential for diagnosis and follow up of patients with retina disorders like macula edema and exudative age-related macular degeneration. Establishing the normal range of central fovea values in our population provides vital baseline data for comparison. Aim: To report the range of normal central fovea thickness measurements in eyes of healthy hospital patients in sub-Saharan Africa using a commercially available Fourier domain optical coherence tomography (OCT) scan. Patients and Methods: A retrospective non-comparative review of case files of a thousand consecutive healthy patients who had retina OCT scans between January 2015 and December 2019 was done. Results: Data from 1000 consecutive eyes of 500 healthy patients were used for the study. There were 181 females and 319 males. The mean central foveal thickness was 239.48 microns (µm), with a minimum thickness of 200.0 µm and maximum thickness of 297.0 µm. Males had significantly (P < 0.001) thicker mean CFT (mean CFT = 241.77 µm) compared with females (mean CFT = 235.43 µm). The mean CFT increased with age of participants by 0.139 µm (P < 0.001) for every year of life below 70. Conclusion: The mean central foveal thickness (CFT) in eyes of healthy patients in our study was 239.48 µm with a range from 200 µm to 297.0 µm. Males had thicker mean CFT compared with females and there was a significant increase in mean CFT by 0.139 µm (P < 0.001) for every year of life below 70.

Capturing fine-grained details for video-based automation of suturing skills assessment.

OBJECTIVES: Manually-collected suturing technical skill scores are strong predictors of continence recovery after robotic radical prostatectomy. Herein, we automate suturing technical skill scoring through computer vision (CV) methods as a scalable method to provide feedback. METHODS: Twenty-two surgeons completed a suturing exercise three times on the Mimic™ Flex VR simulator. Instrument kinematic data (XYZ coordinates of each instrument and pose) were captured at 30 Hz. After standardized training, three human raters manually video segmented suturing task into four sub-stitch phases (Needle handling, Needle targeting, Needle driving, Needle withdrawal) and labeled the corresponding technical skill domains (Needle positioning, Needle entry, Needle driving, and Needle withdrawal). The CV framework extracted RGB features and optical flow frames using a pre-trained AlexNet. Additional CV strategies including auxiliary supervision (using kinematic data during training only) and attention mechanisms were implemented to improve performance. RESULTS: This study included data from 15 expert surgeons (median caseload 300 [IQR 165-750]) and 7 training surgeons (0 [IQR 0-8]). In all, 226 virtual sutures were captured. Automated assessments for Needle positioning performed best with the simplest approach (1 s video; AUC 0.749). Remaining skill domains exhibited improvements with the implementation of auxiliary supervision and attention mechanisms when deployed separately (AUC 0.604-0.794). All techniques combined produced the best performance, particularly for Needle driving and Needle withdrawal (AUC 0.959 and 0.879, respectively). CONCLUSIONS: This study demonstrated the best performance of automated suturing technical skills assessment to date using advanced CV techniques. Future work will determine if a "human in the loop" is necessary to verify surgeon evaluations.

Discovery of putative inhibitors against main drivers of SARS-CoV-2 infection: Insight from quantum mechanical evaluation and molecular modeling.

SARS-CoV-2 triggered a worldwide medical crisis, affecting the world's social, emotional, physical, and economic equilibrium. However, treatment choices and targets for finding a solution to COVID-19's threat are becoming limited. A viable approach to combating the threat of COVID-19 is by unraveling newer pharmacological and therapeutic targets pertinent in the viral survival and adaptive mechanisms within the host biological milieu which in turn provides the opportunity to discover promising inhibitors against COVID-19. Therefore, using high-throughput virtual screening, manually curated compounds library from some medicinal plants were screened against four main drivers of SARS-CoV-2 (spike glycoprotein, PLpro, 3CLpro, and RdRp). In addition, molecular docking, Prime MM/GBSA (molecular mechanics/generalized Born surface area) analysis, molecular dynamics (MD) simulation, and drug-likeness screening were performed to identify potential phytodrugs candidates for COVID-19 treatment. In support of these approaches, we used a series of computational modeling approaches to develop therapeutic agents against COVID-19. Out of the screened compounds against the selected SARS-CoV-2 therapeutic targets, only compounds with no violations of Lipinski's rule of five and high binding affinity were considered as potential anti-COVID-19 drugs. However, lonchocarpol A, diplacol, and broussonol E (lead compounds) were recorded as the best compounds that satisfied this requirement, and they demonstrated their highest binding affinity against 3CLpro. Therefore, the 3CLpro target and the three lead compounds were selected for further analysis. Through protein-ligand mapping and interaction profiling, the three lead compounds formed essential interactions such as hydrogen bonds and hydrophobic interactions with amino acid residues at the binding pocket of 3CLpro. The key amino acid residues at the 3CLpro active site participating in the hydrophobic and polar inter/intra molecular interaction were TYR54, PRO52, CYS44, MET49, MET165, CYS145, HIS41, THR26, THR25, GLN189, and THR190. The compounds demonstrated stable protein-ligand complexes in the active site of the target (3CLpro) over a 100 ns simulation period with stable protein-ligand trajectories. Drug-likeness screening shows that the compounds are druggable molecules, and the toxicity descriptors established that the compounds demonstrated a good biosafety profile. Furthermore, the compounds were chemically reactive with promising molecular electron potential properties. Collectively, we propose that the discovered lead compounds may open the way for establishing phytodrugs to manage COVID-19 pandemics and new chemical libraries to prevent COVID-19 entry into the host based on the findings of this computational investigation.

Oleic acid is an endogenous ligand of TLX/NR2E1 that triggers hippocampal neurogenesis.

Neural stem cells, the source of newborn neurons in the adult hippocampus, are intimately involved in learning and memory, mood, and stress response. Despite considerable progress in understanding the biology of neural stem cells and neurogenesis, regulating the neural stem cell population precisely has remained elusive because we have lacked the specific targets to stimulate their proliferation and neurogenesis. The orphan nuclear receptor TLX/NR2E1 governs neural stem and progenitor cell self-renewal and proliferation, but the precise mechanism by which it accomplishes this is not well understood because its endogenous ligand is not known. Here, we identify oleic acid (18:1ω9 monounsaturated fatty acid) as such a ligand. We first show that oleic acid is critical for neural stem cell survival. Next, we demonstrate that it binds to TLX to convert it from a transcriptional repressor to a transcriptional activator of cell-cycle and neurogenesis genes, which in turn increases neural stem cell mitotic activity and drives hippocampal neurogenesis in mice. Interestingly, oleic acid-activated TLX strongly up-regulates cell cycle genes while only modestly up-regulating neurogenic genes. We propose a model in which sufficient quantities of this endogenous ligand must bind to TLX to trigger the switch to proliferation and drive the progeny toward neuronal lineage. Oleic acid thus serves as a metabolic regulator of TLX activity that can be used to selectively target neural stem cells, paving the way for future therapeutic manipulations to counteract pathogenic impairments of neurogenesis.

Discovery of small molecules targeting the tandem tudor domain of the epigenetic factor UHRF1 using fragment-based ligand discovery.

Despite the established roles of the epigenetic factor UHRF1 in oncogenesis, no UHRF1-targeting therapeutics have been reported to date. In this study, we use fragment-based ligand discovery to identify novel scaffolds for targeting the isolated UHRF1 tandem Tudor domain (TTD), which recognizes the heterochromatin-associated histone mark H3K9me3 and supports intramolecular contacts with other regions of UHRF1. Using both binding-based and function-based screens of a ~ 2300-fragment library in parallel, we identified 2,4-lutidine as a hit for follow-up NMR and X-ray crystallography studies. Unlike previous reported ligands, 2,4-lutidine binds to two binding pockets that are in close proximity on TTD and so has the potential to be evolved into more potent inhibitors using a fragment-linking strategy. Our study provides a useful starting point for developing potent chemical probes against UHRF1.

An ethnobotanical survey of medicinal plants used by traditional healers for the treatment of cancer in Hammanskraal and Winterveld, Tshwane Metropolitan Municipality, South Africa.

Background: Knowledge of medicinal plants used by the traditional healers are mostly confined among the locals and the adherents, hence, proper enquiry and documentation can help the ever dynamic scientific world to find permanent cure to the menace of such deadly diseases such as cancer. This study aimed at (1) specifically recording medicinal plants traditionally used for the treatment of cancer in Hammanskraal and Winterveld, South Africa, and (2) documenting the different methods of preparation and administration of those medicinal plants as recorded during the study. Method: An open-ended semi-structured questionnaire was administered to 90 willing traditional healers in Hammanskraal and Winterveld area, Tshwane Municipality, South Africa to document plants used for cancer treatments. The study was conducted over a period of six months (July - December, 2018). Descriptive statistics was used to present the obtained data. Results: The study recorded twenty-eight plant species belonging to 18 families for the treatment of different types of cancer. Plant species in the Fabaceae family particularly Lessertia frutescens (L.) Goldblatt and J.C. Manning, Senna italica Mill and Trifolium pratense L. were the most prominently mentioned (highest citation frequency) by the traditional healers for lung, and skin cancer treatment. Based on the citation frequency, the most treated cancer by the traditional healers is in the order: skin cancer > lung cancer > breast cancer > prostate cancer > cervical cancer. The method of preparation included decoction (32.3%), infusion (29%), paste (16.1%) and maceration (22.6%). Conclusion: In addition to the documentation of indigenous knowledge related to the use of medicinal plants in the traditional management of cancer in South Africa, this study opens a vista for investigations into the phytochemical and pharmacological properties of the documented plants.

Occurrence of mecA and blaZ genes in methicillin-resistant Staphylococcus aureus associated with vaginitis among pregnant women in Ado-Ekiti, Nigeria.

Methicillin-resistant Staphylococcus aureus (MRSA) is an increasingly prevalent pathogen. We studied the prevalence of MRSA and its association with vaginitis during pregnancy. Bacteriological investigations of high vaginal swabs of 350 healthy pregnant women attending antenatal clinics were carried out. Staphylococci were isolated from high vaginal swabs of 135 of the women. The staphylococcal isolates were resistant to multiple antibiotics. The PCR amplification of DNA of 20 selected isolates yielded six possessing the mecA gene and 13 the blaZ gene. MRSA possessing both the mecA and blaZ genes were isolated from subjects who reported vaginal discharge and itching.

Are outcomes of surgery for retina detachments from giant tears worse than from other holes and tears in this era of small gauge vitrectomy and perflorocarbon use in Sub-Saharan Africa?

AIMS: To report a comparison of clinical features, visual and anatomic outcomes between patients with retinal detachments from giant retinal tears (GRTs) and those from other types of holes and tears undergoing retina reattachment surgery in the same institution within the same time period in sub-Saharan Africa. MATERIALS AND METHODS: A retrospective noncomparative case series of patients undergoing primary retina detachment (RD) repair for rhegmatogenous retina detachment (RRD) at Eye Foundation Hospital Retina Institute between January 2014 and December 2018 was done. RESULTS: A total of 275 eyes of 275 patients met the inclusion criteria. Ages ranged from 7 to 87 years. And 81 (29.4%) eyes had giant tears were categorized as group A and 194 (70.6%) eyes had other types of tears were categorized as group B. At presentation Visual acuity in 66 eyes (79.6%) in the group A was worse than 3/60, compared to 117 eyes (60.3%) in group B. Primary anatomic success was achieved in 73 eyes (92.4%) in-group A and 157 eyes (86.7%) in group B. Final anatomic success was achieved in 75 eyes (94.9%) in group A and 164 eyes (93.2%) in group B. Good visual outcomes were achieved in 48 eyes (59.3%) in group A and 126 eyes (65.6%) in group B. CONCLUSION: Good anatomic and visual outcomes can be achieved after surgery for RRD secondary to giant tears in a sub-Saharan Africa setting in this era of small gauge vitrectomy and perflourocarbon use, these outcomes are comparable to those from surgery for RRD secondary to other types of holes and tears.

Treatment of colorectal cancer in Sub-Saharan Africa: Results from a prospective Nigerian hospital registry.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer worldwide. Mortality for CRC is improving in high income countries, but in low and middle income countries, rates of disease and death from disease are rising. In Sub-Saharan Africa, the ratio of CRC mortality to incidence is the highest in the world. This study investigated the nature of CRC treatment currently being offered and received in Nigeria. METHODS: Between April 2013 and October 2017, a prospective study of consecutively diagnosed cases of CRC was conducted. Patient demographics, clinical features, and treatment recommended and received was recorded for each case. Patients were followed during the study period every 3 months or until death. RESULTS: Three hundred patients were included in our analysis. Seventy-one percent of patients received a recommended surgical operation. Of those that didn't undergo surgery as recommended, 37% cited cost as the main reason, 30% declined due to personal reasons, and less than 5% absconded or were lost to follow up. Approximately half of patients (50.5%) received a chemotherapy regimen when it was recommended, and 4.1% received radiotherapy when this was advised as optimal treatment. With therapy, the median overall survival for patients diagnosed with stage III and stage IV CRC was 24 and 10.5 months respectively. Overall, we found significantly better median survival for patients that received the recommended treatment (25 vs 7 months; P < .01). CONCLUSIONS: A number of patients were unable to receive the recommended treatment, reflecting some of the burden of untreated CRC in the region. Receiving the recommended treatment was associated with a significant difference in outcome. Improved healthcare financing, literacy, training, access, and a better understanding of tumor biology will be necessary to address this discrepancy.

Publisher Correction: Inhibition of TBK1/IKKε Promotes Regeneration of Pancreatic ß-cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Synthesis of Enantiomerically Pure 5-Substituted Piperazine-2-Acetic Acid Esters as Intermediates for Library Production.

The piperazine heterocycle is housed within a large number of FDA-approved drugs and biological probe compounds. Structurally, however, these compounds are mostly confined to substitutions on the two ring nitrogen atoms, rationalizing the expansion of piperazine chemical diversity through carbon substitutions. On the basis of the concept of systematic chemical diversity, a divergent six-step synthesis was developed in which chiral amino acids were transformed, with high diastereoselectivity, into either cis or trans 5-substituted piperazine-2-acetic acid esters that could be chromatographically rendered diastereomerically homogeneous. Starting from six commercially available amino acids or their respective amino alcohols (both antipodes), we obtained a complete set of 24 protected chiral 2,5-disubstituted piperazines, as single stereoisomers in multigram quantities. These diverse and versatile piperazines can be functionalized on either nitrogen atom, allowing them to be used as starting materials for parallel library synthesis and as intermediates for the targeted production of more complex C-substituted piperazine compounds.

Inhibition of TBK1/IKKε Promotes Regeneration of Pancreatic ß-cells.

ß-cell proliferation induction is a promising therapeutic strategy to restore ß-cell mass. By screening small molecules in a transgenic zebrafish model of type 1 diabetes, we identified inhibitors of non-canonical IκB kinases (IKKs), TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε), as enhancers of ß-cell regeneration. The most potent ß-cell regeneration enhancer was a cinnamic acid derivative (E)-3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA), which, acting through the cAMP-dependent protein kinase A (PKA), stimulated ß-cell-specific proliferation by increasing cyclic AMP (cAMP) levels and mechanistic target of rapamycin (mTOR) activity. A combination of PIAA and cilostamide, an inhibitor of ß-cell-enriched cAMP hydrolyzing enzyme phosphodiesterase (PDE) 3, enhanced ß-cell proliferation, whereas overexpression of PDE3 blunted the mitogenic effect of PIAA in zebrafish. PIAA augmented proliferation of INS-1ß-cells and ß-cells in mammalian islets including human islets with elevation in cAMP levels and insulin secretion. PIAA improved glycemic control in streptozotocin (STZ)-induced diabetic mice with increases in ß-cell proliferation, ß-cell area, and insulin content in the pancreas. Collectively, these data reveal an evolutionarily conserved and critical role of TBK1/IKKε suppression in expanding functional ß-cell mass.

Synthesis of Enantiomerically Pure 3-Substituted Piperazine-2-acetic Acid Esters as Intermediates for Library Production.

The piperazine heterocycle is broadly exploited in FDA-approved drugs and biologically active compounds, but its chemical diversity is usually limited to ring nitrogen substitutions, leaving the four carbon atoms underutilized. Using an efficient six-step synthesis, chiral amino acids were transformed into 3-substituted piperazine-2-acetic acid esters as diastereomeric mixtures whose cis and trans products (dr 0.56 → 2.2:1, respectively) could be chromatographically separated. From five amino acids (both antipodes) was obtained a complete matrix of 20 monoprotected chiral 2,3-disubstituted piperazines, each as a single absolute stereoisomer, all but one in multigram quantities. In keeping with our overall purpose of constructing more Csp3-enriched compound libraries for drug discovery, these diverse and versatile piperazines can be functionalized on either nitrogen atom, allowing them to be used as scaffolds for parallel library synthesis and as intermediates for the production of novel piperazine compounds.

The effects of cleaning/disinfecting agents on biogas production in the anaerobic digestion of cow dung slurry.

Cleaning/disinfecting agents (CDA) are compounds known to affect the growth of microbes and could have impacts on biogas production in a digester. In this study, three commonly used CDAs (harpic, hypo and izal) in livestock pens and slaughter houses were applied at different digestion stages and concentrations to cow dung (CD) slurry, with the aim of determining their effects on biogas production. Three application stages (early addition, late addition and graduated addition) were carried out for each CDA, and a CD treatment with no CDA applied was set up as the control. The results showed that application stage had different effects on the parameters of CD monitored during the digestion. Significant (p ≤ .05) effect was recorded on: temperature of izal + CD treatment, pH of harpic + CD and hypo + CD treatments, total bacterial count of hypo + CD and izal + CD treatments and biogas of harpic + CD, hypo + CD and izal + CD treatments. Early addition and increased concentration of hypo appeared toxic to anaerobic bacteria, whereas early addition of izal seemed not to affect their development. While early addition and increased concentration of harpic and hypo inhibited biogas production, the contrary was observed with izal. At the end of digestion, biogas yield (% of control) in the CDA + CD treatments ranged between 8% and 69.4% (harpic + CD), 5.4-23.5% (hypo + CD) and 16.1-52.5% (izal + CD). Among the CDAs used, hypo had the highest inhibitory effect on biogas production from CD slurry. The introduction of harpic at fourth week of digestion showed the least inhibitory effect.

Assessment of trace metals using lichen transplant from automobile mechanic workshop in Ile-Ife metropolis, Nigeria.

The level of air pollution around the automobile mechanic workshops has been generally overlooked. This study, examined the level of trace metals in automobile mechanic workshops and the suitability of using transplanted lichen thalli of Lepraria incana for measuring air pollution in such areas. Samples of the lichen thalli were transplanted into seven different sites and were attached to the bark of trees at each site. The samples were harvested from the sites after 3-month exposure. Concentrations of Pb, Cu, Cd, Fe, Zn, and S content were determined using an atomic absorption spectrophotometer. Results showed that there was a significant difference in the trace metals concentrations across the sites (p < 0.05). The analyzed lichen samples showed a range of 91.26-119.35 ppm for Fe, 30.23-61.32 ppm for Zn, 1.25-2.45 ppm for Cu, 0.017-0.043 ppm for Cd, 0.018-0.051 ppm, and 0.37-0.42 ppm for S. From the study, sites 6 and 7 presented higher concentrations of Cd, Pb, and Zn than other sites. The enrichment factor calculated showed that Zn, Cd, and Pb were greatly enriched from the workshops. The trend in the concentration of these heavy metals suggests that activities in these workshops might become a major source of certain heavy metals in the environment and if the pollution activities persist, it might become worrisome over time.