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.

Plerixafor for pathogen-agnostic treatment in murine thigh infection and zebrafish sepsis.

Plerixafor is a CXCR4 antagonist approved in 2008 by the FDA for hematopoietic stem cell collection. Subsequently, plerixafor has shown promise as a potential pathogen-agnostic immunomodulator in a variety of preclinical animal models. Additionally, investigator-led studies demonstrated plerixafor prevents viral and bacterial infections in patients with WHIM syndrome, a rare immunodeficiency with aberrant CXCR4 signaling. Here, we investigated whether plerixafor could be repurposed to treat sepsis or severe wound infections, either alone or as an adjunct therapy. In a Pseudomonas aeruginosa lipopolysaccharide (LPS)-induced zebrafish sepsis model, plerixafor reduced sepsis mortality and morbidity assessed by tail edema. There was a U-shaped response curve with the greatest effect seen at 0.1 µM concentration. We used Acinetobacter baumannii infection in a neutropenic murine thigh infection model. Plerixafor did not show reduced bacterial growth at 24 h in the mouse thigh model, nor did it amplify the effects of a rifampin antibiotic therapy, in varying regimens. While plerixafor did not mitigate or treat bacterial wound infections in mice, it did reduce sepsis mortality in zebra fish. The observed mortality reduction in our LPS model of zebrafish was consistent with prior research demonstrating a mortality benefit in a murine model of sepsis. However, based on our results, plerixafor is unlikely to be successful as an adjunct therapy for wound infections. Further research is needed to better define the scope of plerixafor as a pathogen-agnostic therapy. Future directions may include the use of longer acting CXCR4 antagonists, biased CXCR4 signaling, and optimization of animal models.

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.

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.

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.

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.

Structure and function of the SIT1 proline transporter in complex with the COVID-19 receptor ACE2.

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.

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.

Semisynthetic derivatives of the fungal metabolite eupenifeldin via targeting the tropolone hydroxy groups.

Eupenifeldin (1) is a fungal secondary metabolite possessing bis-tropolone moieties that demonstrates nanomolar cytotoxic activity against a number of cancer cell types. As a potential anticancer lead, this meroterpenoid was used to access 29 semisynthetic analogues via functionalization of the reactive hydroxy groups of the bis-tropolones. A series of ester (2-6), carbonate (7-8), sulfonate (9-16), carbamate (17-20), and ether (21-30) analogues of 1 were generated via 22 reactions. Most of these compounds were disubstituted, produced via functionalization of both of the tropolonic hydroxy moieties, although three mono-functionalized analogues (6, 8, and 24) and one tri-functionalized analogue (3) were also obtained. The cytotoxic activities of 1-30 were evaluated against human melanoma and ovarian cancer cell lines (i.e., MDA-MB-435 and OVCAR3, respectively). Ester and carbonate analogues of 1 (i.e., 2-8) maintained cytotoxicity at the nanomolar level, and the greatest improvement in aqueous solubility came from the monosuccinate analogue, which was acylated on the secondary hydroxy at the 11 position (6).