Ultrasound-mediated delivery of doxorubicin to the brain results in immune modulation and improved responses to PD-1 blockade in gliomas.

Given the marginal penetration of most drugs across the blood-brain barrier, the efficacy of various agents remains limited for glioblastoma (GBM). Here we employ low-intensity pulsed ultrasound (LIPU) and intravenously administered microbubbles (MB) to open the blood-brain barrier and increase the concentration of liposomal doxorubicin and PD-1 blocking antibodies (aPD-1). We report results on a cohort of 4 GBM patients and preclinical models treated with this approach. LIPU/MB increases the concentration of doxorubicin by 2-fold and 3.9-fold in the human and murine brains two days after sonication, respectively. Similarly, LIPU/MB-mediated blood-brain barrier disruption leads to a 6-fold and a 2-fold increase in aPD-1 concentrations in murine brains and peritumoral brain regions from GBM patients treated with pembrolizumab, respectively. Doxorubicin and aPD-1 delivered with LIPU/MB upregulate major histocompatibility complex (MHC) class I and II in tumor cells. Increased brain concentrations of doxorubicin achieved by LIPU/MB elicit IFN-γ and MHC class I expression in microglia and macrophages. Doxorubicin and aPD-1 delivered with LIPU/MB results in the long-term survival of most glioma-bearing mice, which rely on myeloid cells and lymphocytes for their efficacy. Overall, this translational study supports the utility of LIPU/MB to potentiate the antitumoral activities of doxorubicin and aPD-1 for GBM.

Six-year progression of medically treated infective aortitis.

OBJECTIVE: To report a medically treated case of infective aortitis with mycotic aneurysms that went on to have many years of surveillance imaging. This has not yet been documented as current recommendations for infective aortitis strongly suggest operative intervention combined with aggressive antibiotics, with very high reported mortality for non-operative management. Thus, the natural progression of sac growth during the acute infective period and in the long-term has had an opportunity to be explored. METHODS: A 77-year-old patient presented with infective aortitis confirmed on computed tomography angiography and refused operative intervention despite being explained the associated risks and benefits. She was treated aggressively with antibiotics and monitored in the community, successfully clearing the infection. RESULTS: She received a total of 6 weeks of ceftriaxone intravenously and 1 year of oral ciprofloxacin. She rapidly developed mycotic aneurysmal disease both infrarenal and suprarenal which stabilised within 1 year after diagnosis and did not progress further. CONCLUSIONS: Infective aortitis with mycotic aneurysms is usually treated surgically due to the significant risk of rupture in the acute period. This case suggests that if the acute infective period is passed, the aneurysmal disease stabilises and does not progress.

Brain-wide neuronal circuit connectome of human glioblastoma.

Glioblastoma (GBM), a universally fatal brain cancer, infiltrates the brain and can be synaptically innervated by neurons, which drives tumor progression 1-6 . Synaptic inputs onto GBM cells identified so far are largely short-range and glutamatergic 7-9 . The extent of integration of GBM cells into brain-wide neuronal circuitry is not well understood. Here we applied a rabies virus-mediated retrograde monosynaptic tracing approach 10-12 to systematically investigate circuit integration of human GBM organoids transplanted into adult mice. We found that GBM cells from multiple patients rapidly integrated into brain-wide neuronal circuits and exhibited diverse local and long-range connectivity. Beyond glutamatergic inputs, we identified a variety of neuromodulatory inputs across the brain, including cholinergic inputs from the basal forebrain. Acute acetylcholine stimulation induced sustained calcium oscillations and long-lasting transcriptional reprogramming of GBM cells into a more invasive state via the metabotropic CHRM3 receptor. CHRM3 downregulation suppressed GBM cell invasion, proliferation, and survival in vitro and in vivo. Together, these results reveal the capacity of human GBM cells to rapidly and robustly integrate into anatomically and molecularly diverse neuronal circuitry in the adult brain and support a model wherein rapid synapse formation onto GBM cells and transient activation of upstream neurons may lead to a long-lasting increase in fitness to promote tumor infiltration and progression.

Minimizing pneumocephalus during deep brain stimulation surgery.

BACKGROUND: Deep brain stimulation (DBS) surgery is an effective treatment for movement disorders. Introduction of intracranial air following dura opening in DBS surgery can result in targeting inaccuracy and suboptimal outcomes. We develop and evaluate a simple method to minimize pneumocephalus during DBS surgery. METHODS: A retrospective analysis of prospectively collected data was performed on patients undergoing DBS surgery at our institution from 2014 to 2022. A total of 172 leads placed in 89 patients undergoing awake or asleep DBS surgery were analyzed. Pneumocephalus volume was compared between leads placed with PMT and leads placed with standard dural opening. (112 PMT vs. 60 OPEN). Immediate post-operative high-resolution CT scans were obtained for all leads placed, from which pneumocephalus volume was determined through a semi-automated protocol with ITK-SNAP software. Awake surgery was conducted with the head positioned at 15-30°, asleep surgery was conducted at 0°. RESULTS: PMT reduced pneumocephalus from 11.2 cm3±9.2 to 0.8 cm3±1.8 (P<0.0001) in the first hemisphere and from 7.6 cm3 ± 8.4 to 0.43 cm3 ± 0.9 (P<0.0001) in the second hemisphere. No differences in adverse events were noted between PMT and control cases. Lower rates of post-operative headache were observed in PMT group. CONCLUSION: We present and validate a simple yet efficacious technique to reduce pneumocephalus during DBS surgery.

Highly parallelizable path sampling with minimal rejections using asynchronous replica exchange and infinite swaps.

Capturing rare yet pivotal events poses a significant challenge for molecular simulations. Path sampling provides a unique approach to tackle this issue without altering the potential energy landscape or dynamics, enabling recovery of both thermodynamic and kinetic information. However, despite its exponential acceleration compared to standard molecular dynamics, generating numerous trajectories can still require a long time. By harnessing our recent algorithmic innovations-particularly subtrajectory moves with high acceptance, coupled with asynchronous replica exchange featuring infinite swaps-we establish a highly parallelizable and rapidly converging path sampling protocol, compatible with diverse high-performance computing architectures. We demonstrate our approach on the liquid-vapor phase transition in superheated water, the unfolding of the chignolin protein, and water dissociation. The latter, performed at the ab initio level, achieves comparable statistical accuracy within days, in contrast to a previous study requiring over a year.

PyRETIS 3: Conquering rare and slow events without boundaries.

We present and discuss the advancements made in PyRETIS 3, the third instalment of our Python library for an efficient and user-friendly rare event simulation, focused to execute molecular simulations with replica exchange transition interface sampling (RETIS) and its variations. Apart from a general rewiring of the internal code towards a more modular structure, several recently developed sampling strategies have been implemented. These include recently developed Monte Carlo moves to increase path decorrelation and convergence rate, and new ensemble definitions to handle the challenges of long-lived metastable states and transitions with unbounded reactant and product states. Additionally, the post-analysis software PyVisa is now embedded in the main code, allowing fast use of machine-learning algorithms for clustering and visualising collective variables in the simulation data.

Mismatch repair deficiency is not sufficient to elicit tumor immunogenicity.

DNA mismatch repair deficiency (MMRd) is associated with a high tumor mutational burden (TMB) and sensitivity to immune checkpoint blockade (ICB) therapy. Nevertheless, most MMRd tumors do not durably respond to ICB and critical questions remain about immunosurveillance and TMB in these tumors. In the present study, we developed autochthonous mouse models of MMRd lung and colon cancer. Surprisingly, these models did not display increased T cell infiltration or ICB response, which we showed to be the result of substantial intratumor heterogeneity of mutations. Furthermore, we found that immunosurveillance shapes the clonal architecture but not the overall burden of neoantigens, and T cell responses against subclonal neoantigens are blunted. Finally, we showed that clonal, but not subclonal, neoantigen burden predicts ICB response in clinical trials of MMRd gastric and colorectal cancer. These results provide important context for understanding immune evasion in cancers with a high TMB and have major implications for therapies aimed at increasing TMB.

RNA fusion transcript panel identifies diverse repertoire of fusions in adult glioma patients with therapeutic implications.

Background: Recurrent gliomas are therapeutically challenging diseases with few treatment options available. One area of potential therapeutic vulnerability is the presence of targetable oncogenic fusion proteins. Methods: To better understand the clinical benefit of routinely testing for fusion proteins in adult glioma patients, we performed a retrospective review of 647 adult patients with glioma who underwent surgical resection at our center between August 2017 and May 2021 and whose tumors were analyzed with an in-house fusion transcript panel. Results: Fifty-two patients (8%) were found to harbor a potentially targetable fusion with 11 (21%) of these patients receiving treatment with a fusion-targeted inhibitor. The targetable genes found to be involved in a fusion included FGFR3, MET, EGFR, NTRK1, NTRK2, BRAF, ROS1, and PIK3CA. Conclusions: This analysis demonstrates that routine clinical testing for gene fusions identifies a diverse repertoire of potential therapeutic targets in adult patients with glioma and can offer rational therapeutic options for patients with recurrent disease.

Blue light therapy in the management of chronic wounds: a narrative review of its physiological basis and clinical evidence.

INTRODUCTION: Chronic wounds are a significant problem worldwide, with substantial cost to health care systems; thus, a minimally invasive and well-tolerated treatment is attractive. Blue light has shown promise in wound healing through the principle of photobiomodulation. OBJECTIVE: This review examines the physiological effects of blue light on tissue and the hypothesis that appropriate application of blue light in conjunction with SOC improves wound healing compared with SOC alone. METHODS: The authors searched in PubMed, Google Scholar, and the Cochrane Library to identify literature on the mechanism of action of blue light and then examined the clinical evidence. RESULTS: Key physiological pathways of blue light include generation of ROS and nitric oxide, resulting in promotion of angiogenesis, reduced inflammation, and direct antimicrobial effects. These reactions are seen only at low doses; in fact, higher doses may be harmful to tissue. The only primary study with statistical analyses demonstrated wound area reduction of 51% (P =.007) in blue light-irradiated wounds compared with SOC alone. CONCLUSIONS: Blue light applied following a strict protocol is safe and shows promise in the management of chronic wounds. The current evidence is poor, however, and randomized trials are required to confirm its clinical utility.

Repeated blood-brain barrier opening with an implantable ultrasound device for delivery of albumin-bound paclitaxel in patients with recurrent glioblastoma: a phase 1 trial.

BACKGROUND: Low-intensity pulsed ultrasound with concomitant administration of intravenous microbubbles (LIPU-MB) can be used to open the blood-brain barrier. We aimed to assess the safety and pharmacokinetics of LIPU-MB to enhance the delivery of albumin-bound paclitaxel to the peritumoural brain of patients with recurrent glioblastoma. METHODS: We conducted a dose-escalation phase 1 clinical trial in adults (aged ≥18 years) with recurrent glioblastoma, a tumour diameter of 70 mm or smaller, and a Karnofsky performance status of at least 70. A nine-emitter ultrasound device was implanted into a skull window after tumour resection. LIPU-MB with intravenous albumin-bound paclitaxel infusion was done every 3 weeks for up to six cycles. Six dose levels of albumin-bound paclitaxel (40 mg/m2, 80 mg/m2, 135 mg/m2, 175 mg/m2, 215 mg/m2, and 260 mg/m2) were evaluated. The primary endpoint was dose-limiting toxicity occurring during the first cycle of sonication and albumin-bound paclitaxel chemotherapy. Safety was assessed in all treated patients. Analyses were done in the per-protocol population. Blood-brain barrier opening was investigated by MRI before and after sonication. We also did pharmacokinetic analyses of LIPU-MB in a subgroup of patients from the current study and a subgroup of patients who received carboplatin as part of a similar trial (NCT03744026). This study is registered with ClinicalTrials.gov, NCT04528680, and a phase 2 trial is currently open for accrual. FINDINGS: 17 patients (nine men and eight women) were enrolled between Oct 29, 2020, and Feb 21, 2022. As of data cutoff on Sept 6, 2022, median follow-up was 11·89 months (IQR 11·12-12·78). One patient was treated per dose level of albumin-bound paclitaxel for levels 1 to 5 (40-215 mg/m2), and 12 patients were treated at dose level 6 (260 mg/m2). A total of 68 cycles of LIPU-MB-based blood-brain barrier opening were done (median 3 cycles per patient [range 2-6]). At a dose of 260 mg/m2, encephalopathy (grade 3) occurred in one (8%) of 12 patients during the first cycle (considered a dose-limiting toxicity), and in one other patient during the second cycle (grade 2). In both cases, the toxicity resolved and treatment continued at a lower dose of albumin-bound paclitaxel, with a dose of 175 mg/m2 in the case of the grade 3 encephalopathy, and to 215 mg/m2 in the case of the grade 2 encephalopathy. Grade 2 peripheral neuropathy was observed in one patient during the third cycle of 260 mg/m2 albumin-bound paclitaxel. No progressive neurological deficits attributed to LIPU-MB were observed. LIPU-MB-based blood-brain barrier opening was most commonly associated with immediate yet transient grade 1-2 headache (12 [71%] of 17 patients). The most common grade 3-4 treatment-emergent adverse events were neutropenia (eight [47%]), leukopenia (five [29%]), and hypertension (five [29%]). No treatment-related deaths occurred during the study. Imaging analysis showed blood-brain barrier opening in the brain regions targeted by LIPU-MB, which diminished over the first 1 h after sonication. Pharmacokinetic analyses showed that LIPU-MB led to increases in the mean brain parenchymal concentrations of albumin-bound paclitaxel (from 0·037 µM [95% CI 0·022-0·063] in non-sonicated brain to 0·139 µM [0·083-0·232] in sonicated brain [3·7-times increase], p<0·0001) and carboplatin (from 0·991 µM [0·562-1·747] in non-sonicated brain to 5·878 µM [3·462-9·980] µM in sonicated brain [5·9-times increase], p=0·0001). INTERPRETATION: LIPU-MB using a skull-implantable ultrasound device transiently opens the blood-brain barrier allowing for safe, repeated penetration of cytotoxic drugs into the brain. This study has prompted a subsequent phase 2 study combining LIPU-MB with albumin-bound paclitaxel plus carboplatin (NCT04528680), which is ongoing. FUNDING: National Institutes of Health and National Cancer Institute, Moceri Family Foundation, and the Panattoni family.

Initiating a Magnetic Resonance-Guided Focused Ultrasound Program: Comprehensive Workflow and Lessons Learned from the Initial 116 Cases.

INTRODUCTION: Magnetic resonance-guided focused ultrasound (MRgFUS) represents an incisionless treatment option for essential or parkinsonian tremor. The incisionless nature of this procedure has garnered interest from both patients and providers. As such, an increasing number of centers are initiating new MRgFUS programs, necessitating development of unique workflows to optimize patient care and safety. Herein, we describe establishment of a multi-disciplinary team, workflow processes, and outcomes for a new MRgFUS program. METHODS: This is a single-academic center retrospective review of 116 consecutive patients treated for hand tremor between 2020 and 2022. MRgFUS team members, treatment workflow, and treatment logistics were reviewed and categorized. Tremor severity and adverse events were evaluated at baseline, 3, 6, and 12 months post-MRgFUS with the Clinical Rating Scale for Tremor Part B (CRST-B). Trends in outcome and treatment parameters over time were assessed. Workflow and technical modifications were noted. RESULTS: The procedure, workflow, and team members remained consistent throughout all treatments. Technique modifications were attempted to reduce adverse events. A significant reduction in CRST-B score was achieved at 3 months (84.5%), 6 months (79.8%), and 12 months (72.2%) post-procedure (p < 0.0001). The most common post-procedure adverse events in the acute period (<1 day) were gait imbalance (61.1%), fatigue and/or lethargy (25.0%), dysarthria (23.2%), headache (20.4%), and lip/hand paresthesia (13.9%). By 12 months, the majority of adverse events had resolved with a residual 17.8% reporting gait imbalance, 2.2% dysarthria, and 8.9% lip/hand paresthesia. No significant trends in treatment parameters were found. CONCLUSIONS: We demonstrate the feasibility of establishing an MRgFUS program with a relatively rapid increase in evaluation and treatment of patients while maintaining high standards of safety and quality. While efficacious and durable, adverse events occur and can be permanent in MRgFUS.

Checkpoint kinase 1/2 inhibition potentiates anti-tumoral immune response and sensitizes gliomas to immune checkpoint blockade.

Whereas the contribution of tumor microenvironment to the profound immune suppression of glioblastoma (GBM) is clear, tumor-cell intrinsic mechanisms that regulate resistance to CD8 T cell mediated killing are less understood. Kinases are potentially druggable targets that drive tumor progression and might influence immune response. Here, we perform an in vivo CRISPR screen to identify glioma intrinsic kinases that contribute to evasion of tumor cells from CD8 T cell recognition. The screen reveals checkpoint kinase 2 (Chek2) to be the most important kinase contributing to escape from CD8 T-cell recognition. Genetic depletion or pharmacological inhibition of Chek2 with blood-brain-barrier permeable drugs that are currently being evaluated in clinical trials, in combination with PD-1 or PD-L1 blockade, lead to survival benefit in multiple preclinical glioma models. Mechanistically, loss of Chek2 enhances antigen presentation, STING pathway activation and PD-L1 expression in mouse gliomas. Analysis of human GBMs demonstrates that Chek2 expression is inversely associated with antigen presentation and T-cell activation. Collectively, these results support Chek2 as a promising target for enhancement of response to immune checkpoint blockade therapy in GBM.

Path sampling with memory reduction and replica exchange to reach long permeation timescales.

Assessing kinetics in biological processes with molecular dynamics simulations remains a computational and conceptual challenge, given the large time and length scales involved. For kinetic transport of biochemical compounds or drug molecules, the permeability through the phospholipid membranes is a key kinetic property, but long timescales are hindering the accurate computation. Technological advances in high-performance computing therefore need to be accompanied by theoretical and methodological developments. In this contribution, the replica exchange transition interface sampling (RETIS) methodology is shown to give perspective toward observing longer permeation pathways. It is first reviewed how RETIS, a path-sampling methodology that gives in principle exact kinetics, can be used to compute membrane permeability. Next, recent and current developments in three RETIS aspects are discussed: several new Monte Carlo moves in the path-sampling algorithm, memory reduction by reducing pathlengths, and exploitation of parallel computing with CPU-imbalanced replicas. Finally, the memory reduction presenting a new replica exchange implementation, coined REPPTIS, is showcased with a permeant needing to pass a membrane with two permeation channels, either representing an entropic or energetic barrier. The REPPTIS results showed clearly that inclusion of some memory and enhancing ergodic sampling via replica exchange moves are both necessary to obtain correct permeability estimates. In an additional example, ibuprofen permeation through a dipalmitoylphosphatidylcholine membrane was modeled. REPPTIS succeeded in estimating the permeability of this amphiphilic drug molecule with metastable states along the permeation pathway. In conclusion, the presented methodological advances allow for deeper insight into membrane biophysics even if the pathways are slow, as RETIS and REPPTIS push the permeability calculations to longer timescales.

Epitranscriptomic regulation of cortical neurogenesis via Mettl8-dependent mitochondrial tRNA m3C modification.

Increasing evidence implicates the critical roles of various epitranscriptomic RNA modifications in different biological processes. Methyltransferase METTL8 installs 3-methylcytosine (m3C) modification of mitochondrial tRNAs in vitro; however, its role in intact biological systems is unknown. Here, we show that Mettl8 is localized in mitochondria and installs m3C specifically on mitochondrial tRNAThr/Ser(UCN) in mouse embryonic cortical neural stem cells. At molecular and cellular levels, Mettl8 deletion in cortical neural stem cells leads to reduced mitochondrial protein translation and attenuated respiration activity. At the functional level, conditional Mettl8 deletion in mice results in impaired embryonic cortical neural stem cell maintenance in vivo, which can be rescued by pharmacologically enhancing mitochondrial functions. Similarly, METTL8 promotes mitochondrial protein expression and neural stem cell maintenance in human forebrain cortical organoids. Together, our study reveals a conserved epitranscriptomic mechanism of Mettl8 and mitochondrial tRNA m3C modification in maintaining embryonic cortical neural stem cells in mice and humans.

Enhanced path sampling using subtrajectory Monte Carlo moves.

Path sampling allows the study of rare events, such as chemical reactions, nucleation, and protein folding, via a Monte Carlo (MC) exploration in path space. Instead of configuration points, this method samples short molecular dynamics (MD) trajectories with specific start- and end-conditions. As in configuration MC, its efficiency highly depends on the types of MC moves. Since the last two decades, the central MC move for path sampling has been the so-called shooting move in which a perturbed phase point of the old path is propagated backward and forward in time to generate a new path. Recently, we proposed the subtrajectory moves, stone-skipping (SS) and web-throwing, that are demonstrably more efficient. However, the one-step crossing requirement makes them somewhat more difficult to implement in combination with external MD programs or when the order parameter determination is expensive. In this article, we present strategies to address the issue. The most generic solution is a new member of subtrajectory moves, wire fencing (WF), that is less thrifty than the SS but more versatile. This makes it easier to link path sampling codes with external MD packages and provides a practical solution for cases where the calculation of the order parameter is expensive or not a simple function of geometry. We demonstrate the WF move in a double-well Langevin model, a thin film breaking transition based on classical force fields, and a smaller ruthenium redox reaction at the ab initio level in which the order parameter explicitly depends on the electron density.

Management of pediatric appendicitis during the COVID-19 pandemic: A nationwide multicenter cohort study.

BACKGROUND: The COVID-19 pandemic has impacted timely access to care for children, including patients with appendicitis. This study aimed to evaluate the effect of the COVID-19 pandemic on management of appendicitis and patient outcomes. METHODS: A multicenter retrospective study was performed including 19 children's hospitals from April 2019-October 2020 of children (age≤18 years) diagnosed with appendicitis. Groups were defined by each hospital's city/state stay-at-home orders (SAHO), designating patients as Pre-COVID (Pre-SAHO) or COVID (Post-SAHO). Demographic, treatment, and outcome data were obtained, and univariate and multivariable analysis was performed. RESULTS: Of 6,014 patients, 2,413 (40.1%) presented during the COVID-19 pandemic. More patients were managed non-operatively during the COVID-19 pandemic compared to before the pandemic (147 (6.1%) vs 144 (4.0%), p < 0.001). Despite this change, there was no difference in the proportion of complicated appendicitis between groups (1,247 (34.6%) vs 849 (35.2%), p = 0.12). COVID era non-operative patients received fewer additional procedures, including interventional radiology (IR) drain placements, compared to pre-COVID non-operative patients (29 (19.7%) vs 69 (47.9%), p < 0.001). On adjusted analysis, factors associated with increased odds of receiving non-operative management included: increasing duration of symptoms (OR=1.01, 95% CI: 1.01-1.012), African American race (OR=2.4, 95% CI: 1.3-4.6), and testing positive for COVID-19 (OR=10.8, 95% CI: 5.4-21.6). CONCLUSION: Non-operative management of appendicitis increased during the COVID-19 pandemic. Additionally, fewer COVID era cases required IR procedures. These changes in the management of pediatric appendicitis during the COVID pandemic demonstrates the potential for future utilization of non-operative management.

Glioblastoma modeling with 3D organoids: progress and challenges.

Glioblastoma (GBM) is the most aggressive adult primary brain tumor with nearly universal treatment resistance and recurrence. The mainstay of therapy remains maximal safe surgical resection followed by concurrent radiation therapy and temozolomide chemotherapy. Despite intensive investigation, alternative treatment options, such as immunotherapy or targeted molecular therapy, have yielded limited success to achieve long-term remission. This difficulty is partly due to the lack of pre-clinical models that fully recapitulate the intratumoral and intertumoral heterogeneity of GBM and the complex tumor microenvironment. Recently, GBM 3D organoids originating from resected patient tumors, genetic manipulation of induced pluripotent stem cell (iPSC)-derived brain organoids and bio-printing or fusion with non-malignant tissues have emerged as novel culture systems to portray the biology of GBM. Here, we highlight several methodologies for generating GBM organoids and discuss insights gained using such organoid models compared to classic modeling approaches using cell lines and xenografts. We also outline limitations of current GBM 3D organoids, most notably the difficulty retaining the tumor microenvironment, and discuss current efforts for improvements. Finally, we propose potential applications of organoid models for a deeper mechanistic understanding of GBM and therapeutic development.

Exchanging Replicas with Unequal Cost, Infinitely and Permanently.

We developed a replica exchange method that is effectively parallelizable even if the computational cost of the Monte Carlo moves in the parallel replicas are considerably different, for instance, because the replicas run on different types of processor units or because of the algorithmic complexity. To prove detailed-balance, we make a paradigm shift from the common conceptual viewpoint in which the set of parallel replicas represents a high-dimensional superstate, to an ensemble-based criterion in which the other ensembles represent an environment that might or might not participate in the Monte Carlo move. In addition, based on a recent algorithm for computing permanents, we effectively increase the exchange rate to infinite without the steep factorial scaling as a function of the number of replicas. We illustrate the effectiveness of this replica exchange methodology by combining it with a quantitative path sampling method, replica exchange transition interface sampling (RETIS), in which the costs for a Monte Carlo move can vary enormously as paths in a RETIS algorithm do not have the same length and the average path lengths tend to vary considerably for the different path ensembles that run in parallel. This combination, coined ∞RETIS, was tested on three model systems.