Revolutionizing DNA repair research and cancer therapy with CRISPR-Cas screens.

All organisms possess molecular mechanisms that govern DNA repair and associated DNA damage response (DDR) processes. Owing to their relevance to human disease, most notably cancer, these mechanisms have been studied extensively, yet new DNA repair and/or DDR factors and functional interactions between them are still being uncovered. The emergence of CRISPR technologies and CRISPR-based genetic screens has enabled genome-scale analyses of gene-gene and gene-drug interactions, thereby providing new insights into cellular processes in distinct DDR-deficiency genetic backgrounds and conditions. In this Review, we discuss the mechanistic basis of CRISPR-Cas genetic screening approaches and describe how they have contributed to our understanding of DNA repair and DDR pathways. We discuss how DNA repair pathways are regulated, and identify and characterize crosstalk between them. We also highlight the impacts of CRISPR-based studies in identifying novel strategies for cancer therapy, and in understanding, overcoming and even exploiting cancer-drug resistance, for example in the contexts of PARP inhibition, homologous recombination deficiencies and/or replication stress. Lastly, we present the DDR CRISPR screen (DDRcs) portal , in which we have collected and reanalysed data from CRISPR screen studies and provide a tool for systematically exploring them.

Decitabine cytotoxicity is promoted by dCMP deaminase DCTD and mitigated by SUMO-dependent E3 ligase TOPORS.

The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA-protein crosslinks (DNMT1-DPCs), leading to DNA hypomethylation. However, 5-aza-dC's clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with the identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment.

Effect of an Antacid (Aluminum Hydroxide/Magnesium Hydroxide/Simethicone) or a Proton Pump Inhibitor (Omeprazole) on the Pharmacokinetics of Tebipenem Pivoxil Hydrobromide (TBP-PI-HBr) in Healthy Adult Subjects.

Tebipenem pivoxil hydrobromide (TBP-PI-HBr) is a novel oral carbapenem prodrug being developed for the treatment of serious bacterial infections. This open-label, 3-period, fixed sequence study evaluated the effect of gastric acid-reducing agents, aluminum hydroxide/magnesium hydroxide/simethicone, and omeprazole on the pharmacokinetics (PK) of tebipenem (TBP), the active moiety, following coadministration with immediate release TBP-PI-HBr during fasting. In Period 1, subjects received a single oral dose of TBP-PI-HBr 600 mg (2 × 300 mg tablets). In Period 2, subjects received a single oral dose of aluminum hydroxide 800 mg/magnesium hydroxide 800 mg/simethicone 80 mg suspension co-administered with a single dose of TBP-PI-HBr 600 mg. In Period 3, subjects received a single oral dose of omeprazole 40 mg once daily over 5 days, followed by single dose administration of TBP-PI-HBr 600 mg on day 5. In each period, whole blood samples were obtained prior to, and up to 24 h, following TBP-PI-HBr dose administration in order to characterize TBP PK. A 7-day washout was required between periods. Twenty subjects were enrolled and completed the study. Following co-administration of TBP-PI-HBr with either aluminum hydroxide/magnesium hydroxide/simethicone or omeprazole, total TBP exposure (area under the curve [AUC]) was approximately 11% (geometric mean ratio 89.2, 90% confidence interval: 83,2, 95.7) lower, and Cmax was 22% (geometric mean ratio 78.4, 90% confidence interval: 67.9, 90.6) and 43% (geometric mean ratio 56.9, 90% confidence interval: 49.2, 65.8) lower, respectively, compared to administration of TBP-PI-HBr alone. Mean TBP elimination half-life (t1/2) was generally comparable across treatments (range: 1.0 to 1.5 h). Concomitant administration of TBP-PI-HBr with omeprazole or aluminum hydroxide/magnesium hydroxide/simethicone is not expected to impact the efficacy of TBP-PI-HBr, as there is minimal impact on TBP plasma AUC, which is the pharmacodynamic driver of efficacy. Co-administration was generally safe and well tolerated.

Absorption, Metabolism, and Excretion of [14C]-Tebipenem Pivoxil Hydrobromide (TBP-PI-HBr) in Healthy Male Subjects.

Tebipenem pivoxil hydrobromide (TBP-PI-HBr) is an oral prodrug of pharmacologically active moiety tebipenem (TBP), which is a carbapenem with activity against multidrug-resistant Gram-negative pathogens. Conversion from the prodrug to the active moiety, namely, TBP, occurs in the enterocytes of the gastrointestinal tract via intestinal esterases. The absorption, metabolism, and excretion in humans were evaluated, following the administration of a single oral dose of [14C]-TBP-PI-HBr. Healthy male subjects (n = 8) received a single 600 mg oral dose of TBP-PI-HBr containing approximately 150 µCi of [14C]-TBP-PI-HBr. Blood, urine, and fecal samples were collected to determine the total radioactivity, concentrations of TBP (plasma only), and metabolite profiling and identification. The overall mean recovery of the total radioactivity in urine (38.7%) and feces (44.6%) combined was approximately 83.3% of the administered dose, with individual recoveries ranging from 80.1% to 85.0%. Plasma TBP LC-MS/MS and metabolite profiling data suggest that TBP was the main circulating component in plasma and that it accounts for approximately 54% of the total plasma radioactivity, based on the plasma AUC ratio of TBP/total radioactivity. The ring-open metabolite LJC 11562 was another major component in plasma (>10%). TBP (M12), LJC 11562, and four trace to minor metabolites were identified/characterized in the urine. TBP-PI, TBP (M12), and 11 trace to minor metabolites were identified/characterized in the feces. The renal and fecal routes are major clearance pathways in the elimination of [14C]-TBP-PI-HBr, with a mean combined recovery of 83.3%. TBP and its inactive ring-open metabolite LJC 11562 were the major circulating metabolites in the plasma.

Impact of antibiotic pharmacokinetics in urine on recurrent bacteriuria following treatment of complicated urinary tract infections.

The clinical relevance of bacteriuria following antibiotic treatment of complicated urinary tract infections in clinical trials remains controversial. We evaluated the impact of urine pharmacokinetics on the timing of recurrent bacteriuria in a recently completed trial that compared oral tebipenem pivoxil hydrobromide to intravenous ertapenem. The urinary clearance and urine dwell time of ertapenem were prolonged relative to tebipenem and were associated with a temporal difference in the repopulation of bladder urine with bacteria following treatment, potentially confounding the assessment of efficacy.

A thread-based electrofluidic platform for direct transfer, separation, and pre-concentration of materials from sample swabs.

A new method and platform has been developed for direct transfer, electrophoretic separation, and pre-concentration of swabbed samples using the principles of thread-based electrofluidics. A direct electrokinetic injection has been observed for a variety of analytes ranging from small molecules to proteins. The effect of physicochemical interactions of the analyte with the swab and the thread on the transfer efficiency has been studied by exploring different swab and thread combinations. For fluorescein, using a polyurethane swab, 98% and 94% transfer efficiencies were observed on mercerised cotton and nylon thread, while only 80% transfer efficiency was observed on polyester thread, respectively. A 97% transfer of fluorescein was observed on the nylon thread when a flocked nylon swab was used, while only 47% transfer was observed when a cotton swab was used. A successful transfer has been observed for both liquid and dry samples from either pre-wetted or dry swabs in both the presence and absence of any surrounding electrolytes. The platform has been further adapted for multiplexed analysis, where a sample from a single swab was transferred onto two parallel thread systems with ca. 50% distribution between them. The method has been validated for transfer, separation, and pre-concentration of DNA from blood. It has also been successfully used to directly analyse dried blood samples using a commercial sampling device, Neoteryx Mitra.

Recent advances in miniaturization of portable liquid chromatography with emphasis on detection.

Liquid chromatography is a prominent analytical technique in separation science and chemical analysis, applied across numerous fields of research and within industrial applications. Over the past few decades, there has been a growing interest in the miniaturization of this technique, which has been particularly enabled through new miniature and portable detection technologies for in-field, at-site, and point-of-need (collectively 'out-of-lab') analyses. Accordingly, significant advances have been made in recent years in the development of miniaturized liquid chromatography with photometric, electrochemical, and mass spectrometric detection, enabling the development of field-deployable and portable instruments for various applications. Herein, recent developments in the miniaturization of detection systems for inclusion within, and/or coupling with, portable liquid chromatographic systems, are reviewed in detail together with critical comments and expected future trends in this area.

Plasma and Intrapulmonary Concentrations of Tebipenem following Oral Administration of Tebipenem Pivoxil Hydrobromide to Healthy Adult Subjects.

Tebipenem pivoxil hydrobromide (TBP-PI-HBr) is an oral carbapenem prodrug being developed for the treatment of serious bacterial infections. The active moiety, tebipenem, has broad-spectrum activity against common Enterobacterales pathogens, including extended-spectrum-ß-lactamase (ESBL)-producing multidrug-resistant strains. This study evaluated the intrapulmonary pharmacokinetics (PK) and epithelial lining fluid (ELF) and alveolar macrophage (AM) concentrations of tebipenem relative to plasma levels in nonsmoking, healthy adult subjects. Thirty subjects received oral TBP-PI-HBr at 600 mg every 8 h for five doses. Serial blood samples were collected following the last dose. Each subject underwent one standardized bronchoscopy with bronchoalveolar lavage (BAL) 1, 2, 4, 6, or 8 h after the fifth dose of TBP-PI-HBr. The tebipenem area under the concentration-time curve for the 8-h dosing interval (AUC0-8) values in plasma, ELF, and AMs were calculated using the mean concentration at each BAL sampling time. Ratios of AUC0-8 values for total ELF and AMs to those for unbound plasma were determined, using a plasma protein binding value of 42%. Mean values ± standard deviations (SD) of tebipenem maximum (Cmax) and minimum (Cmin) total plasma concentrations were 11.37 ± 3.87 mg/L and 0.043 ± 0.039 mg/L, respectively. Peak tebipenem concentrations in plasma, ELF, and AMs occurred at 1 h and then decreased over 8 h. Ratios of tebipenem AUC0-8 values for ELF and AMs to those for unbound plasma were 0.191 and 0.047, respectively. Four (13.3%) subjects experienced adverse events (diarrhea, fatigue, papule, and coronavirus disease 2019 [COVID-19]); all resolved, and none were severe or serious. Tebipenem is distributed into the lungs of healthy adults, which supports the further evaluation of TBP-PI-HBr for the treatment of lower respiratory tract bacterial infections caused by susceptible pathogens. (This study has been registered at ClinicalTrials.gov under identifier NCT04710407.).

Pharmacokinetics of Oral Tebipenem Pivoxil Hydrobromide in Subjects with Various Degrees of Renal Impairment.

Tebipenem pivoxil hydrobromide (TBP-PI-HBr) is an oral carbapenem prodrug antimicrobial agent with broad-spectrum activity that includes multidrug-resistant (MDR) Enterobacterales. This study evaluated the safety, tolerability, and pharmacokinetics of TBP-PI-HBr in healthy subjects with normal renal function (cohort 1) and subjects with various degrees of renal impairment (RI [cohorts 2 to 4]) or end-stage renal disease (ESRD) receiving hemodialysis (HD) (cohort 5). Subjects in cohorts 1 to 4 received a single oral dose of TBP-PI-HBr (600 mg). Subjects in cohort 5 received single-dose administration (600 mg) in 2 separate periods: pre-HD (period 2) and post-HD (period 1). Pharmacokinetic (PK) parameters for TBP, the active moiety, were determined using noncompartmental analysis. Compared with cohort 1, the TBP plasma area under the curve (AUC) increased 1.4- to 4.5-fold among cohorts 2 to 4, the maximum concentration of drug in plasma (Cmax) increased up to 1.3-fold and renal clearance (CLR) decreased from 13.4 L/h to 2.4 L/h as the severity of RI increased. Plasma TBP concentrations decreased over 8 to 12 h in cohorts 1 to 4, and apparent total body clearance (CL/F) correlated (R2 = 0.585) with creatinine clearance (CLCR). TBP urinary excretion ranged from 38% to 64% of the administered dose for cohorts 1 to 4. Subjects in cohort 5 had an approximately 7-fold increase in TBP AUC and elimination half-life (t1/2) versus cohort 1. After 4 h of HD, mean TBP plasma exposure decreased by approximately 40%. Overall, TBP plasma exposure increased with increasing RI, highlighting the renal route importance in TBP elimination. A dose reduction of TBP-PI-HBr may be needed in patients with RI (CLCR of ≤50 mL/min) and those with ESRD on HD. TBP-PI-HBr was well tolerated across all cohorts. (This study has been registered at ClinicalTrials.gov under registration no. NCT04178577.).

Single- and Multiple-Ascending-Dose Study of the Safety, Tolerability, and Pharmacokinetics of the Polymyxin Derivative SPR206.

Carbapenem-resistant Acinetobacter baumannii and Enterobacterales are identified as urgent threats, and multidrug-resistant (MDR) Pseudomonas aeruginosa and extended-spectrum beta-lactamase (ESBL)-producing pathogens are identified as serious threats by the Centers for Disease Control and Prevention (CDC). SPR206 is a novel polymyxin derivative with potent in vitro and in vivo activity against A. baumannii, P. aeruginosa, and multiple clinically important species of Enterobacterales, including multidrug- and extensively drug-resistant strains. This was a first-in-human (FIH) double-blind, placebo-controlled, single-, and multiple-ascending-dose study of the safety, tolerability, and pharmacokinetics (PK) of SPR206 in 94 healthy subjects. Following intravenous (i.v.) administration (1-h infusion) at single doses of 10 mg to 400 mg and multiple doses of 25 mg to 150 mg every 8 h (q8h) for 7 days and 100 mg q8h for 14 days, SPR206 was generally safe and generally well tolerated. While the incidence of adverse events increased with dose, most were of mild severity. Systemic exposure (maximum concentration of drug in serum [Cmax] and area under the concentration-time curve [AUC]) to SPR206 was approximately dose proportional, time to peak concentrations ranged from 1.1 to 1.3 h, and half-life ranged from 2.4 to 4.1 h. No appreciable accumulation occurred with repeated dosing of SPR206, and trough concentrations suggest that steady state was achieved by day 2. Urinary excretion of unchanged SPR206 was dose dependent across single- (SAD) and multiple-ascending-dose (MAD) cohorts, and the percentage of dose excreted as SPR206 was up to >50%. Importantly, no evidence of nephrotoxicity was observed over 14 days of 100 mg q8h dosing of SPR206; a dosing regimen anticipated to exceed requirements for clinical efficacy. (This study has been registered at ClinicalTrials.gov under identifier NCT03792308.).

First-in-Human Evaluation of the Safety, Tolerability, and Pharmacokinetics of SPR720, a Novel Oral Bacterial DNA Gyrase (GyrB) Inhibitor for Mycobacterial Infections.

SPR720 (phosphate prodrug of SPR719) is a novel aminobenzimidazole bacterial DNA gyrase (GyrB) inhibitor in development for nontuberculous mycobacterial pulmonary disease (NTM-PD) and pulmonary tuberculosis. SPR719 has demonstrated activity against clinically relevant mycobacteria in vitro and in murine and hollow-fiber infection models. This phase 1 randomized, double-blind, placebo-controlled, single ascending dose (SAD)/multiple ascending dose (MAD) trial evaluated the safety, tolerability, and pharmacokinetics of SPR720/SPR719. A total of 96 healthy volunteers (n = 8/cohort, 3:1 randomization) received SPR720 (or placebo) as single oral doses ranging from 100 to 2,000 mg or repeat total daily doses ranging from 500 to 1,500 mg for 7 or 14 days. SPR720 was well tolerated at daily doses of up to 1,000 mg for up to 14 days. Across SAD/MAD cohorts, the most common adverse events (AEs) were gastrointestinal (nausea, vomiting, and diarrhea) and headache, all of mild or moderate severity and dose dependent. No serious AEs were reported. The median SPR719 Tmax ranged from 2.8 to 8.0 h across cohorts, and the t1/2 ranged from 2.9 to 4.5 h and was shown to be dose independent. Dosing with food decreased SPR719 plasma exposure by approximately 20%. In the MAD cohorts, SPR719 plasma exposure declined approximately 40% between days 1 and 7, suggesting induction of an elimination pathway. However, plasma AUC0-24 was comparable between days 7 and 14. The results of this first-in-human study suggest that predicted therapeutic exposures of SPR719 can be attained with a once-daily oral administration of SPR720. (This study has been registered at ClinicalTrials.gov under registration no. NCT03796910.).

Randomized, Double-Blind, Placebo- and Positive-Controlled Crossover Study of the Effects of Tebipenem Pivoxil Hydrobromide on QT/QTc Intervals in Healthy Subjects.

Tebipenem pivoxil hydrobromide (TBP-PI-HBr) is an orally available prodrug of tebipenem (TBP), a carbapenem with in vitro activity against multidrug-resistant Gram-negative pathogens. This study evaluated the effects of single therapeutic and supratherapeutic doses of TBP-PI-HBr on the heart rate-corrected QT interval (QTc) by assessing the concentration-QT interval relationship using exposure-response modeling. This was a randomized, double-blind, placebo- and active-controlled, single-dose, four-way crossover study. Subjects received single oral doses of TBP-PI-HBr at 600 and 1,200 mg, placebo, and positive control (moxifloxacin at 400 mg). Cardiodynamic electrocardiograms (ECGs) and blood samples were collected in each period. Twenty-four subjects were enrolled. TBP-PI-HBr had no clinically significant adverse effects on heart rate or ECG parameters. The model-predicted slope suggests that the baseline-corrected difference in heart rate from placebo was not importantly affected by plasma TBP concentrations, supporting the use of the QT interval corrected by Fridericia's method as an appropriate correction. The model-predicted difference in QTc at the mean maximum concentration (Cmax) for TBP had negative predicted values for each dose, and no QTc prolongation was detected following TBP-PI-HBr at 600 mg or 1,200 mg. Assay sensitivity was established with moxifloxacin at 400 mg. Exposure to TBP increased in a dose-dependent manner with 600- and 1,200-mg doses. The TBP area under the concentration-time curve from time zero to infinity and Cmax with the 1,200-mg dose were 1.8- and 1.3-fold greater, respectively, than those with the 600-mg dose. TBP-PI-HBr was generally safe and well tolerated, with no effect in QT interval prolongation.

Small-Footprint, Field-Deployable LC/MS System for On-Site Analysis of Per- and Polyfluoroalkyl Substances in Soil.

Per- and polyfluoroalkyl substances (PFASs) are emerging environmental pollutants of global concern. For rapid field site evaluation, there are very few sensitive, field-deployable analytical techniques. In this work, a portable lightweight capillary liquid chromatography (capLC) system was coupled with a small footprint portable mass spectrometer and configured for field-based applications. Further, an at-site ultrasound-assisted extraction (pUAE) methodology was developed and applied with a portable capLC/mass spectrometry (MS) system for on-site analysis of PFASs in real soil samples. The influential variables on the integration of capLC with MS and on the resolution and signal intensity of the capLC/MS setup were investigated. The important parameters affecting the efficiency of the pUAE method were also studied and optimized using the response surface methodology based on a central composite design. The mean recovery for 11 PFASs ranged between 70 and 110%, with relative standard deviations ranging from 3 to 12%. In-field method sensitivity for 12 PFASs ranged from 0.6 to 0.1 ng/g, with wide dynamic ranges (1-600 ng/g) and excellent linearities (R2 > 0.991). The in-field portable system was benchmarked against a commercial lab-based LC-tandem MS (MS/MS) system for the analysis of PFASs in real soil samples, with the results showing good agreement. When deployed to a field site, 12 PFASs were detected and identified in real soil samples at concentrations ranging from 8.1 ng/g (for perfluorooctanesulfonic acid) to 2935.0 ng/g (perfluorohexanesulfonic acid).

Miniature Multiwavelength Deep UV-LED-Based Absorption Detection System for Capillary LC.

A new miniature deep UV absorbance detector has been developed using low-cost and high-performance LEDs, which can be operated in both scanning (230 to 300 nm) and individual wavelength (240, 255, and 275 nm) detection modes. The detector is mostly composed of off-the-shelf components, such as LEDs, trifurcated fiber optic assembly, a capillary Z-type flow cell, and photodiodes. It has been characterized for use with a standard capillary LC system and was benchmarked against a standard variable wavelength capillary LC detector. The detector shows very low levels of stray light (<0.4%), utilization of up to 99.0% of the effective path length of the flow cell, a wide dynamic range (0.5 to 200 µg/mL for sulfamethazine, carbamazepine, and flavone), and low noise levels (at 300 µAU level). The detector was applied within a miniaturized LC system.

Epidemiology and Outcomes of Early Morning Neuroparalytic Syndrome Following Snake Bite-A Retrospective Study.

OBJECTIVE: Snake envenomation has been poorly studied in developing countries. 'Early morning neuroparalytic syndrome' (EMNS), the classical clinical constellation caused by krait bites, refers to nighttime, indoor bites where nonspecific symptoms progress to neuroparalysis. Literature regarding EMNS in children is scarce. This study was planned to describe the clinical profile, intensive care needs and predictors of outcome in children with EMNS. METHODS: It is a retrospective study of children below 12 years admitted with a clinical diagnosis of snake envenomation to the pediatric intensive care unit (PICU) of a tertiary care hospital in North India. Patient records were reviewed from the electronic patient database manager. Comparison was made between the EMNS group and the non-EMNS group and between survivors and nonsurvivors within the EMNS group. RESULTS: Of the 111 children with snake envenomation, 76 had neuroparalysis (68%) and 51 had EMNS. In the EMNS cohort, 37 (72.5%) belonged to rural areas, 46 (90.2%) had indoor bites and 39 (76.5%) were witnessed. Patients with EMNS were more likely to have absent fang marks, hypoxemia at admission, bulbar palsy and need for PICU admission. Mortality rate was 13.7% in EMNS; predictors included younger age, presence of ptosis, cardiac arrest at admission and nonavailability of PICU bed (univariable analysis) but none of them independently predicted mortality. CONCLUSION: Younger age, presence of ptosis, cardiac arrest at admission and nonavailability of intensive care beds increase the risk of mortality in children with EMNS. Timely recognition and respiratory support may reduce mortality in these children.

3D Printed Liquid Cooling Interface for a Deep-UV-LED-Based Flow-Through Absorbance Detector.

Ultraviolet (UV)-light-emitting diodes (LEDs) are now widely used in analytical absorbance-based detectors; as compared to conventional UV lamps, they offer lower cost, faster response time, and higher photon conversion efficiency. However, current generation deep-UV-LEDs produce excess heat when operated at normal operating currents, which affects output stability and reduces their overall performance and lifespan. Herein a 3D printed liquid cooling interface has been developed for a deep-UV-LED-based optical detector, for capillary format flow-through detection. The interface consists of a circular channel that is tightly wrapped around the LED to provide active liquid cooling. The design also facilitates easy plug-and-play assembly of the various essential components of the detector: specifically, a 255 nm UV-LED, a capillary Z-cell, and a broadband UV photodiode (PD). The unique liquid cooling interface improved the performance of the detector by reducing the LED temperature up to 22 °C, increasing the spectral output up to 34%, decreasing the required stabilization time by up to 6-fold, and reducing the baseline noise and limits of detection (LODs) by a factor of 2. The detector was successfully used within a capillary HPLC system and could offer a miniaturized, rapidly stabilized, highly sensitive, and low-cost alternative to conventional UV detectors.

Trade-off between synergy and efficacy in combinations of HIV-1 latency-reversing agents.

Eradicating HIV-1 infection is difficult because of the reservoir of latently infected cells that gets established soon after infection, remains hidden from antiretroviral drugs and host immune responses, and retains the capacity to reignite infection following the cessation of treatment. Drugs called latency-reversing agents (LRAs) are being developed to reactivate latently infected cells and render them susceptible to viral cytopathicity or immune killing. Whereas individual LRAs have failed to induce adequate reactivation, pairs of LRAs have been identified recently that act synergistically and hugely increase reactivation levels compared to individual LRAs. The maximum synergy achievable with LRA pairs is of clinical importance, as it would allow latency-reversal with minimal drug exposure. Here, we employed stochastic simulations of HIV-1 transcription and translation in latently infected cells to estimate this maximum synergy. We incorporated the predominant mechanisms of action of the two most promising classes of LRAs, namely, protein kinase C agonists and histone deacetylase inhibitors, and quantified the activity of individual LRAs in the two classes by mapping our simulations to corresponding in vitro experiments. Without any adjustable parameters, our simulations then quantitatively captured experimental observations of latency-reversal when the LRAs were used in pairs. Performing simulations representing a wide range of drug concentrations, we estimated the maximum synergy achievable with these LRA pairs. Importantly, we found with all the LRA pairs we considered that concentrations yielding the maximum synergy did not yield the maximum latency-reversal. Increasing concentrations to increase latency-reversal compromised synergy, unravelling a trade-off between synergy and efficacy in LRA combinations. The maximum synergy realizable with LRA pairs would thus be restricted by the desired level of latency-reversal, a constrained optimum we elucidated with our simulations. We expect this trade-off to be important in defining optimal LRA combinations that would maximize synergy while ensuring adequate latency-reversal.

Investigating the Effect of Column Geometry on Separation Efficiency using 3D Printed Liquid Chromatographic Columns Containing Polymer Monolithic Phases.

Effect of column geometry on the liquid chromatographic separations using 3D printed liquid chromatographic columns with in-column polymerized monoliths has been studied. Three different liquid chromatographic columns were designed and 3D printed in titanium as 2D serpentine, 3D spiral, and 3D serpentine columns, of equal length and i.d. Successful in-column thermal polymerization of mechanically stable poly(BuMA-co-EDMA) monoliths was achieved within each design without any significant structural differences between phases. Van Deemter plots indicated higher efficiencies for the 3D serpentine chromatographic columns with higher aspect ratio turns at higher linear velocities and smaller analysis times as compared to their counterpart columns with lower aspect ratio turns. Computational fluid dynamic simulations of a basic monolithic structure indicated 44%, 90%, 100%, and 118% higher flow through narrow channels in the curved monolithic configuration as compared to the straight monolithic configuration at linear velocities of 1, 2.5, 5, and 10 mm s-1, respectively. Isocratic RPLC separations with the 3D serpentine column resulted in an average 23% and 245% (8 solutes) increase in the number of theoretical plates as compared to the 3D spiral and 2D serpentine columns, respectively. Gradient RPLC separations with the 3D serpentine column resulted in an average 15% and 82% (8 solutes) increase in the peak capacity as compared to the 3D spiral and 2D serpentine columns, respectively. Use of the 3D serpentine column at a higher flow rate, as compared to the 3D spiral column, provided a 58% reduction in the analysis time and 74% increase in the peak capacity for the isocratic separations of the small molecules and the gradient separations of proteins, respectively.

Endovascular reconstruction of aneurysms with a complex geometry.

Conventional endovascular coiling remains the mainstay of treatment for most aneurysms; however, it may not be suitable for aneurysms with a complex geometry and there remains the risk of recanalization. Aneurysms with an unfavorable morphology are difficult to treat through both endovascular and surgical means. Progress in endovascular technology has allowed for the emergence of newer strategies to treat aneurysms with a complex geometry. Better packing density in wide-necked and large aneurysms can be achieved through the balloon remodeling technique. Similarly, a self-expanding stent cannot only act as a scaffold that helps to retain coils but also aids in diverting the blood flow away from the aneurysm sac. Lately, focus has shifted from endosaccular occlusion to endoluminal reconstruction; flow diverters are being increasingly used to treat aneurysms with an unfavorable geometry. However, there is no clear consensus on the best endovascular management strategy in certain subset of aneurysms - large and giant internal carotid aneurysms, blister aneurysms, and fusiform/dissecting aneurysms of the vertebrobasilar artery. We present a review of literature and discuss the current evidence for the various endovascular strategies to treat complex aneurysms.

Scaling law characterizing the dynamics of the transition of HIV-1 to error catastrophe.

Increasing the mutation rate, µ of viruses above a threshold, µ(c) has been predicted to trigger a catastrophic loss of viral genetic information and is being explored as a novel intervention strategy. Here, we examine the dynamics of this transition using stochastic simulations mimicking within-host HIV-1 evolution. We find a scaling law governing the characteristic time of the transition: τ ≈ 0.6/(µ - µ(c)). The law is robust to variations in underlying evolutionary forces and presents guidelines for treatment of HIV-1 infection with mutagens. We estimate that many years of treatment would be required before HIV-1 can suffer an error catastrophe.