Occurrence of "Natural Selection" in Successful Small Molecule Drug Discovery.

Published compounds from ChEMBL version 32 are used to seek evidence for the occurrence of "natural selection" in drug discovery. Three measures of natural product (NP) character were applied, to compare time- and target-matched compounds reaching the clinic (clinical compounds in phase 1-3 development and approved drugs) with background compounds (reference compounds). Pseudo-NPs (PNPs), containing NP fragments combined in ways inaccessible by nature, are increasing over time, reaching 67% of clinical compounds first disclosed since 2010. PNPs are 54% more likely to be found in post-2008 clinical versus reference compounds. The majority of target classes show increased clinical compound NP character versus their reference compounds. Only 176 NP fragments appear in >1000 clinical compounds published since 2008, yet these make up on average 63% of the clinical compound's core scaffolds. There is untapped potential awaiting exploitation, by applying nature's building blocks─"natural intelligence"─to drug design.

Oxygenated versus non-oxygenated flush out during deceased donor liver procurement: The first proof-of-concept study in humans.

BACKGROUND: Liver transplantation is used for treating end-stage liver disease, fulminant hepatitis, and oncological malignancies and organ shortage is a major limiting factor worldwide. The use of grafts based on extended donor criteria have become internationally accepted. Oxygenated machine perfusion technologies are the most recent advances in organ transplantation; however, it is only applied after a period of cold ischemia. Due to its high cost, we aimed to use a novel device, OxyFlush®, based on oxygenation of the preservation solution, applied during liver procurement targeting the maintenance of ATP during static cold storage (SCS). METHODS: Twenty patients were randomly assigned to the OxyFlush or control group based on a 1:1 ratio. In the OxyFlush group, the perfusion solution was oxygenated with OxyFlush® device while the control group received a non-oxygenated solution. Liver and the common bile duct (CBD) biopsies were obtained at three different time points. The first was at the beginning of the procedure, the second during organ preparation, and the third after total liver reperfusion. Biopsies were analyzed, and adenosine triphosphate (ATP) levels and histological scores of the liver parenchyma and CBD were assessed. Postoperative laboratory tests were performed. RESULTS: OxyFlush® was able to maintain ATP levels during SCS and improved the damage caused by the lack of oxygen in the CBD. However, OxyFlush® did not affect laboratory test results and histological findings of the parenchyma. CONCLUSION: We present a novel low-cost device that is feasible and could represent a valuable tool in organ preservation during SCS.

Enhancing the dielectric constant of zwitterionic liquids via dipole moment and anion chemistry.

The dielectric constant is a critical parameter in many energy-related applications. Typically, increasing the dielectric constant of soft materials involves adding high dielectric constant polar liquids or inorganic fillers, but there are limitations to this approach due to safety concerns with volatile and flammable solvents and the agglomeration of inorganic fillers. An alternative approach is to add zwitterionic liquids that exhibit exceptionally high dielectric constants with negligible volatility. Here, we report the synthesis of a series of zwitterionic liquids containing an imidazolium cation, exhibiting the highest dielectric constant among all organic molecules (∼350 at 293 K). The cation-anion linkage was tailored in a wide range between three and nine carbons, rendering the zwitterion dipole from 25 to 52 D. Comparing the dielectric constant for zwitterions with different anions (i.e., sulfonylimide, sulfonate, and carboxylate) reveals the beneficial impacts of the delocalized sulfonylimide anion vs the carboxylate anion due to the enlarged molecular dipole and more homogenous liquid morphology. Molecular dipole and liquid morphology are identified as the keys to developing high dielectric constant zwitterionic liquids. The extremely high dielectric constant accessible with the proposed molecular design paves new avenues for developing high dielectric constant zwitterions that act as dielectricizers.

Investigating Photoactive Antimicrobials as Alternatives (or Adjuncts) to Traditional Therapy.

Photodynamic therapy (PDT) is an established therapy used for the treatment of cutaneous skin cancers and other non-infective ailments. There has been recent interest in the opportunity to use aPDT (antimicrobial PDT) to treat skin and soft tissue infections. PDT utilizes photosensitizers that infiltrate all cells and "sensitize" them to a given wavelength of light. The photosensitizer is simply highly absorbent to a given wavelength of light and when excited will produce, in the presence of oxygen, damaging oxygen radicals and singlet oxygen. Bacterial cells are comparatively poor at combatting oxidative stress when compared with human cells therefore a degree of selective toxicity can be achieved with aPDT.In this chapter, we outline methodologies for testing aPDT in vitro using standard lab equipment.

Investigating Combination Therapy as a Means to Enhance Activity and Repurpose Antimicrobials.

Current clinical practice assumes that a single antibiotic given as a bolus or as a course will successfully treat most infections. In modern medicine, this is becoming less and less true with drug-resistant, multi-drug-resistant, extensively drug-resistant, and untreatable infections becoming more common. Where single-drug therapy (monotherapy) fails, we will turn to multi-drug therapy. Alternatively, combination therapy could be useful to prevent the emergence of resistance. Multi-drug therapy is already standard for some multi-drug resistant infections and is the standard for the treatment of some pathogens such as Mycobacterium tuberculosis.The use of combination therapy for everyday infections could be a clear course out of the current AMR crisis we are facing. With every additional drug added to a combination (n + 1) the likelihood of the pathogen evolving resistance drops exponentially.Many generic antibiotics are cheap to manufacture as they have fallen out of patent protection but are less effective at pharmacologically effective doses due to overuse in the past. Combination therapy can combine these generic compounds into cocktails that can not only treat susceptible and resistant infections but can also reduce the risk of new resistances arising and can resuscitate the use of antimicrobials once thought defunct.In this chapter, we will summarize theory behind combination therapy and standard in vitro methodologies used.

Monitoring Live Mycobacteria in Real-Time Using a Microfluidic Acoustic-Raman Platform.

Tuberculosis (TB) is the most common cause of death from an infectious disease. Although treatment has been available for more than 70 years, it still takes too long and many patients default risking relapse and the emergence of resistance. It is known that lipid-rich, phenotypically antibiotic-tolerant, bacteria are more resistant to antibiotics and may be responsible for relapse necessitating extended therapy. Using a microfluidic system that acoustically traps live mycobacteria, M. smegmatis, a model organism for M. tuberculosis we can perform optical analysis in the form of wavelength-modulated Raman spectroscopy (WMRS) on the trapped organisms. This system can allow observations of the mycobacteria for up to 8 h. By adding antibiotics, it is possible to study the effect of antibiotics in real-time by comparing the Raman fingerprints in comparison to the unstressed condition. This microfluidic platform may be used to study any microorganism and to dynamically monitor its response to many conditions including antibiotic stress, and changes in the growth media. This opens the possibility of understanding better the stimuli that trigger the lipid-rich downregulated and phenotypically antibiotic-resistant cell state.

Using Hollow Fiber to Model Treatment of Antimicrobial-Resistant Organisms.

The use of animal models is still widespread in science but there is a movement away from this manner of experimentation. One option approved by the FDA for human-like studies is the hollow fiber bioreactor (HFS). HFSs are highly controllable, self-contained systems that allow for the modeling of individual tissues and disease phenotypes. Oxygen, drug concentration & half-life, and immune cell invasion are all scalable to human and veterinary conditions using a HFS. There are drawbacks to the systems including cost and contamination so the use of these systems must be carefully managed.With these limitations in mind, the scope of the technology is great. Antimicrobial susceptibility testing (AST) is possible with greater accuracy and clinical validity than classical in vitro techniques making minimal inhibitory concentration (MIC) data generated on the bench more translatable to the clinic.In this chapter, we will outline the background of the HFS and some typical uses.

The Risk of Microbial Transmission in Recipients of Donor Livers That Underwent Hypothermic or Normothermic Machine Perfusion.

Background: Ex situ machine perfusion is increasingly used to preserve and assess donor livers before transplantation. Compared with traditional static cold storage (SCS), machine perfusion exposes livers to an additional risk of microbial contamination. However, information on the risk of microbial transmission during machine perfusion is lacking. Methods: All livers that underwent either hypothermic oxygenated machine perfusion (HOPE) or normothermic machine perfusion (NMP) in our center between September 2021 and September 2023, and during which samples were taken from SCS fluid and/or machine perfusion solution for microbiological examination, were included in this retrospective, observational clinical study. Microbial transmission was examined from SCS fluid to machine perfusion solution fluid and, subsequently, to recipients of these livers. Results: A total of 90 cases of liver machine perfusion were included: 59 HOPE and 31 NMP. SCS preservation fluid cultures before HOPE or NMP were positive for at least 1 microorganism in 52% of the cases. After HOPE, there were no cases of positive machine perfusion fluid or evidence of microbial transmission to the recipients. After NMP, in 1 (3%) patient Escherichia coli was grown from abdominal drain fluid, the same bacterial strain that was also grown from the SCS preservation fluid before NMP. This E coli was resistant to the antibiotics that are routinely added to the NMP perfusion fluid. Conclusions: The risk of microbial transmission after machine perfusion is very low but not absent. We recommend routine sampling of machine perfusion fluid at the end of the procedure for microbiological analysis.

Hydroxyurea to prevent brain injury in children with sickle cell disease (HU Prevent)-A randomized, placebo-controlled phase II feasibility/pilot study.

Central nervous system (CNS) injury is common in sickle cell disease (SCD) and occurs early in life. Hydroxyurea is safe and efficacious for treatment of SCD, but high-quality evidence from randomized trials to estimate its neuroprotective effect is scant. HU Prevent was a randomized (1:1), double-blind, phase II feasibility/pilot trial of dose-escalated hydroxyurea vs. placebo for the primary prevention of CNS injury in children with HbSS or HbS-ß0-thalassemia subtypes of SCD age 12-48 months with normal neurological examination, MRI of the brain, and cerebral blood flow velocity. We hypothesized that hydroxyurea would reduce by 50% the incidence of CNS injury. Two outcomes were compared: primary-a composite of silent cerebral infarction, elevated cerebral blood flow velocity, transient ischemic attack, or stroke; secondary-a weighted score estimating the risk of suffering the consequences of stroke (the Stroke Consequences Risk Score-SCRS), based on the same outcome events. Six participants were randomized to each group. One participant in the hydroxyurea group had a primary outcome vs. four in the placebo group (incidence rate ratio [90% CI] 0.216 [0.009, 1.66], p = .2914) (~80% reduction in the hydroxyurea group). The mean SCRS score was 0.078 (SD 0.174) in the hydroxyurea group, 0.312 (SD 0.174) in the placebo group, p = .072, below the p-value of .10 often used to justify subsequent phase III investigations. Serious adverse events related to study procedures occurred in 3/41 MRIs performed, all related to sedation. These results suggest that hydroxyurea may have profound neuroprotective effect in children with SCD and support a definitive phase III study to encourage the early use of hydroxyurea in all infants with SCD.

Assaying Lysosomal Enzyme Activity in Dictyostelium discoideum.

Lysosomes are membrane-enclosed organelles that digest intracellular material. They contain more than 50 different enzymes that can degrade a variety of macromolecules including nucleic acids, proteins, polysaccharides, and lipids. In addition to functioning within lysosomes, lysosomal enzymes are also secreted. Alterations in the levels and activities of lysosomal enzymes dysregulates lysosomes, which can lead to the intralysosomal accumulation of biological material and the development of lysosomal storage diseases (LSDs) in humans. Dictyostelium discoideum has a long history of being used to study the trafficking and functions of lysosomal enzymes. More recently, it has been used as a model system to study several LSDs. In this chapter, we outline the methods for assessing the activity of several lysosomal enzymes in D. discoideum (α-galactosidase, ß-galactosidase, α-glucosidase, ß-glucosidase, ß-N-acetylglucosaminidase, α-mannosidase, cathepsin B, cathepsin D, cathepsin F, palmitoyl protein thioesterase 1, and tripeptidyl peptidase 1).

Assessing the Benefits and Harms of Pharmacotherapy in Older Adults with Frailty: Insights from Pharmacoepidemiologic Studies of Routine Health Care Data.

The objective of this review is to summarize and appraise the research methodology, emerging findings, and future directions in pharmacoepidemiologic studies assessing the benefits and harms of pharmacotherapies in older adults with different levels of frailty. Older adults living with frailty are at elevated risk for poor health outcomes and adverse effects from pharmacotherapy. However, current evidence is limited due to the under-enrollment of frail older adults and the lack of validated frailty assessments in clinical trials. Recent advancements in measuring frailty in administrative claims and electronic health records (database-derived frailty scores) have enabled researchers to identify patients with frailty and to evaluate the heterogeneity of treatment effects by patients' frailty levels using routine health care data. When selecting a database-derived frailty score, researchers must consider the type of data (e.g., different coding systems), the length of the predictor assessment period, the extent of validation against clinically validated frailty measures, and the possibility of surveillance bias arising from unequal access to care. We reviewed 13 pharmacoepidemiologic studies published on PubMed from 2013 to 2023 that evaluated the benefits and harms of cardiovascular medications, diabetes medications, anti-neoplastic agents, antipsychotic medications, and vaccines by frailty levels. These studies suggest that, while greater frailty is positively associated with adverse treatment outcomes, older adults with frailty can still benefit from pharmacotherapy. Therefore, we recommend routine frailty subgroup analyses in pharmacoepidemiologic studies. Despite data and design limitations, the findings from such studies may be informative to tailor pharmacotherapy for older adults across the frailty spectrum.

Potentiation of BKCa channels by cystic fibrosis transmembrane conductance regulator (CFTR) correctors VX-445 and VX-121.

Cystic fibrosis (CF) results from mutations in the CFTR anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The BKCa potassium channel is also critical for maintaining lung ASL volume. Here, we show the CFTR corrector, VX-445 (Elexacaftor), a component of Trikafta, induces K+ secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BKCa antagonist paxilline. Similar results were observed with VX-121 - a corrector under clinical evaluation. Whole-cell patch-clamp recordings confirmed potentiated channel activity from CFTR correctors on the BKCa α-subunit, and excised patch-clamp recordings demonstrated a significant increase in open probability. In mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced action potential firing frequency in primary hippocampal and cortical neurons. VX-445 effects were observed at low micomolar concentrations (1-10 µM) - within the range reported in plasma and tissues from CF patients. We raise the possibilities that CFTR correctors gain additional clinical benefit by activation of BKCa in the lung, yet may lead to adverse events through BKCa activation, elsewhere.

Neurosurgical Management of Patients with Alpha-Gal Syndrome.

Alpha-gal syndrome (AGS) is an immunoglobulin E-mediated hypersensitivity to galatcose-alpha-1,3-galactose (alpha-gal), a carbohydrate compound present in nonprimate mammalian products. Initial exposure to alpha-gal most often occurs through a tick bite, most commonly the lone star tick in the United States. Repeated exposure to alpha-gal may elicit severe allergic reactions, including anaphylaxis. The allergy restricts dietary intake and may significantly impact perioperative risk, as many medications, anesthetics, and intraoperative surgical products utilize bovine or porcine-derived agents, including those containing magnesium stearate, glycerol, and gelatin. Here, we review the perineurosurgical care of two individuals with AGS and highlight pertinent clinical practices and perioperative management of these patients.

Generalized Hailey-Hailey disease associated with c.2395C>T ATP2C1 gene mutation and fatal outcome.

Hailey-Hailey disease (HHD) is a rare, autosomal dominant genodermatosis caused by a mutation of the ATP2C1 gene and presenting as an erosive dermatosis, particularly in the intertriginous areas. Generalized HHD is a rare variant. We present a case of widespread, recalcitrant HHD in a middle-aged woman with a fatal outcome. No other underlying dermatosis was identified, with the possible exception of drug sensitivity to carbamazepine. Diagnosis of HHD was confirmed by histology and genetic studies which showed a c.2395C>T mutation in the ATP2C1 gene. Concurrent pemphigus was excluded. Cases of generalized HHD are extremely rare and present a challenge in diagnosis and management. Increased awareness of this severe clinical variant is needed to improve quality of care for patients with this form of HHD.

Comparing deposition characteristics of various embolic particles using an in vitro prostate microvasculature model.

PURPOSE: To compare spatial distributions of radiopaque glass (RG) microspheres, trisacryl gelatin (TAG) microspheres, and polyvinyl alcohol (PVA) foam particles within a planar in vitro microvascular model of the hyperplastic hemiprostate. MATERIALS AND METHODS: A microvascular model simulating hyperplastic hemiprostate was perfused with a water-glycerin mixture. A microcatheter was positioned distal to the model's prostatic artery origin and embolic particles (RG: 50 µm, 100 µm, and 150 µm; TAG: 100-300 µm and 300-500 µm; and PVA: 90-180 µm and 180-300 µm) were administered using a syringe pump. Microscopic imaging and subsequent semantic segmentation were performed to quantify particle distributions within the models. Distal penetrations were quantified statistically via modal analysis of the particle distributions. RESULTS: Maximum distal penetration was observed for RG 50, followed by RG 100 and then TAG 100-300 and RG 150. TAG 300-500, PVA 90-180, and PVA 180-300 particles exhibited the lowest distal penetrations. The distal penetration metrics between groups were significantly different (p < 0.05) except between TAG 100-300 and RG 150 and between PVA 90-180 and PVA 180-300. CONCLUSIONS: Comparing the spatial distributions of embolic particles in an in vitro microvascular model simulating the hyperplastic hemiprostate revealed that noncompressible particles and those with narrower size calibrations and smaller relative diameters exhibited higher degrees of distal packing. The embolization front was less distinct for particles with wider size calibrations, which resulted in smaller, more distal emboli along with larger, more proximal emboli. PVA and TAG 300-500 particles both exhibited relatively low overall distal penetration.

Mechanical or Biological Prosthesis for Aortic Valve Replacement in Patients Aged 45 to 74 Years.

OBJECTIVE: The selection of valve prostheses for patients undergoing surgical aortic valve replacement (SAVR) remains controversial. In this study, we compared the long-term outcomes of patients undergoing aortic valve replacement with biological or mechanical aortic valve prostheses. METHODS: We evaluated late results among 5,762 patients aged 45-74 years who underwent biological or mechanical aortic valve replacement with or without concomitant coronary artery bypass from 1989 to 2019 at four medical centers. The Cox proportional hazards model was used to compare late survival; the age-dependent effect of prosthesis type on long-term survival was evaluated by an interaction term between age and prosthesis type. Incidences of stroke, major bleeding, and reoperation on the aortic valve following the index procedure were compared between prosthesis groups. RESULTS: Overall, 61% (n=3,508) of patients received a bioprosthesis. The 30-day mortality rate was 1.7% (n=58) in the bioprosthesis group and 1.5% (n=34) in the mechanical group (P=0.75). During a mean follow-up of 9.0 years, the adjusted risk of mortality was higher in the bioprosthesis group (HR=1.30, P<0.001). The long-term survival benefit associated with mechanical prosthesis persisted until 70 years of age. Bioprosthesis (vs mechanical prosthesis) was associated with a similar risk of stroke (P=0.20), lower risk of major bleeding (P<0.001), and higher risk of reoperation (P<0.001). CONCLUSIONS: Compared to bioprostheses, mechanical aortic valves are associated with a lower adjusted risk of long-term mortality in patients aged 70 years or younger. Patients <70 years old undergoing SAVR should be informed of the potential survival benefit of mechanical valve substitutes.

Durable complete response in a patient with leptomeningeal melanoma after treatment with dabrafenib, trametinib, and nivolumab.

Leptomeningeal disease (LMD) is a devastating complication of melanoma with a dismal prognosis. We present the case of a young man with stage IV BRAF V600E mutant melanoma with lung, lymph node, and brain metastases initially treated with ipilimumab and nivolumab, who subsequently developed LMD. Upon change to BRAF/MEK targeted therapy with nivolumab, a durable complete response was achieved and remains ongoing, off treatment, 7 years from diagnosis. Management of symptomatic LMD remains a critical unmet clinical challenge, with limited clinical trial data. This exceptional case is instructive, as the first published case of the use of the triplet, and the first durable response with therapy discontinuation, in melanoma LMD. The triple-drug regimen may be considered a viable option in fit patients. This case highlights the potential for long-term disease control and the critical and urgent need to develop clinical trials inclusive of patients with LMD to define the best treatment strategies.

ACG Clinical Guideline: Focal Liver Lesions.

Focal liver lesions (FLLs) have become an increasingly common finding on abdominal imaging, especially asymptomatic and incidental liver lesions. Gastroenterologists and hepatologists often see these patients in consultation and make recommendations for management of multiple types of liver lesions, including hepatocellular adenoma, focal nodular hyperplasia, hemangioma, and hepatic cystic lesions including polycystic liver disease. Malignancy is important to consider in the differential diagnosis of FLLs, and healthcare providers must be familiar with the diagnosis and management of FLLs. This American College of Gastroenterology practice guideline uses the best evidence available to make diagnosis and management recommendations for the most common FLLs.

Mono-allelic epigenetic regulation of bi-directional polycistronic transcription initiation by RNA Polymerase II in Trypanosoma brucei.

Unique for a eukaryote, protein-coding genes in trypanosomes are arranged in polycistronic units (PTUs). This genome arrangement has led to a model where Pol II transcription of PTUs is unregulated. The initial step in trypanosome lytic factor (TLF) mediated lysis of Trypanosoma brucei requires high affinity haptoglobin/hemoglobin receptor (HpHbR) binding. Here we demonstrate that by in vitro selection with TLF, resistance is obtained in a stepwise process correlating with loss of HpHbR expression at an allelic level. RNA-seq, Pol II ChIP and run-on analysis indicate HpHbR silencing is at the transcriptional level, where loss of Pol II binding at the promoter region specifically shuts down transcription of the HpHbR containing gene cluster and the adjacent opposing gene cluster. Reversible transcriptional silencing of the divergent PTUs correlates with DNA base J modification of the shared promoter region. Therefore, epigenetic mechanisms exist to regulate gene expression via Pol II transcription initiation of gene clusters in a mono-allelic fashion. These findings suggest epigenetic chromatin-based regulation of gene expression is deeply conserved among eukaryotes, including primitive eukaryotes that rely on polycistronic transcription.