Immune checkpoint inhibitors in high-grade upper tract urothelial carcinoma: Paradigm shift emphasizing organ preservation.

Objective: The aim was to evaluate the role of immune check point inhibitors (ICIs) in patients with high-grade upper tract urothelial carcinoma (UTUC) who are managed endoscopically when nephroureterectomy (NU) is not feasible, such as in patients who are either not candidates for NU or decline extirpative surgery. Methods: All patients diagnosed with high-grade UTUC and managed endoscopically between January 1996 and August 2022 were included in the study. Subsequently, patients were categorised based on their use of ICIs into group 1 (patients who did not receive ICIs) and group 2 (patients who received ICIs). Survival outcomes were assessed using Kaplan-Meier analysis, while a multivariable regression model was employed to analyse the impact of clinical characteristics on survival. Results: A total of 29 patients were enrolled, with 14 in group 1 and 15 in group 2. Both groups exhibited similar demographic and disease characteristics, including multifocality, laterality and initial tumour size. The median follow-up period was 29.2 months. Notably, group 2 demonstrated significantly enhanced overall and metastasis-free survival rates compared to group 1. At 47.8 months, the overall survival rate was 0% (all patients died) in group 1, whereas it was 85.7% in group 2. Similarly, the metastasis-free survival rate was 0% (all patients had metastatic disease) in group 1 at 40.6 months, whereas it reached 78.0% in group 2. The multivariable analysis indicated a correlation between ICI usage and improved survival outcomes, with a hazard ratio of 0.002. Conclusion: Utilisation of adjuvant ICIs in the setting of endoscopically treated patients with high-grade UTUC is associated with significantly improved survival rates. ICIs should be considered in this patient population, however, more studies with larger sample size are warranted.

The relationship between the force applied and perceived by the surgeon during ureteral access sheath placement: ex-vivo experimental model.

PURPOSE: To define a peak force of insertion (PFOI) threshold for ureteral damage during ureteral access sheath (UAS) placement on an experimental ureteral orifice model. METHODS: A specially designed water tank using 2 laparoscopic 5 mm ports and 2 different size (10 Fr and 8 Fr) sealing cap adaptors (SCA) as ureteral orifices was used to perform the test. A 10-12 Fr UAS was fixed to a load cell and the force of insertion (FOI) was continuously recorded with a digital force gauge.13 experts in the field of endourology who participated performed 3 UAS insertions. The FOI was recorded initially with 10 Fr followed by 8 Fr SCA. On the final insertion, the orifice was obstructed, leaving a 5 cm length to insert the UAS. The experts were asked to "Stop at the point they anticipate ureteral damage, and they would not proceed in real life". RESULTS: Using 10 Fr SCA the PFOI was 2.12 ± 0.58 Newton (N) (range:1.48-3.48) while 8 Fr SCA showed a PFOI 5.76 ± 0.96 N (range:4.05-7.35). Six of the experts, said they would stop proceeding when they reached above 5.1 N. Three experts had PFOI < 5.1 N and the other 4 stated they would go with PFOIs of 5.88, 6.16, 6.69 and 7.35 N when using SCA of 8 Fr.The highest load they would stop proceeding had a PFOI of 6.09 ± 1.87 N (range: 2.53-10.74). CONCLUSION: The PFOI threshold for ureteral damage inserting UAS of the experts is variable. Although FOI is a subjective perception, experience suggests that ureteral injury may occur at an average of 6.05 N perceived by surgeons' tactile feedback. In-vivo measurement of UAS PFOI may confirm a threshold.

Using a Function-First "Scout Fragment"-Based Approach to Develop Allosteric Covalent Inhibitors of Conformationally Dynamic Helicase Mechanoenzymes.

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as translation, ribosome assembly, and genome maintenance. Helicases with essential functions in certain cancer cells have been identified, and helicases expressed by many viruses are required for their pathogenicity. Therefore, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop chemical inhibitors for helicases, enzymes with high conformational dynamics. We envisioned that electrophilic "scout fragments", which have been used in chemical proteomic studies, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest an approach to discover covalent inhibitor starting points and druggable allosteric sites in conformationally dynamic mechanoenzymes.

Using a function-first 'scout fragment'-based approach to develop allosteric covalent inhibitors of conformationally dynamic helicase mechanoenzymes.

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as genome replication and maintenance, ribosome assembly and translation. Helicases with essential functions only in certain cancer cells have been identified and helicases expressed by certain viruses are required for their pathogenicity. As a result, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop selective chemical inhibitors for helicases, enzymes with highly dynamic conformations. We envisioned that electrophilic 'scout fragments', which have been used for chemical proteomic based profiling, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest a covalent inhibitor discovery approach to target helicases and potentially other conformationally dynamic mechanoenzymes.

The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation.

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.

A chemical genetics approach to examine the functions of AAA proteins.

The structural conservation across the AAA (ATPases associated with diverse cellular activities) protein family makes designing selective chemical inhibitors challenging. Here, we identify a triazolopyridine-based fragment that binds the AAA domain of human katanin, a microtubule-severing protein. We have developed a model for compound binding and designed ASPIR-1 (allele-specific, proximity-induced reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation. Only in cells expressing mutant katanin does ASPIR-1 treatment increase the accumulation of CAMSAP2 at microtubule minus ends, confirming specific on-target cellular activity. Importantly, ASPIR-1 also selectively inhibits engineered cysteine mutants of human VPS4B and FIGL1-AAA proteins, involved in organelle dynamics and genome stability, respectively. Structural studies confirm our model for compound binding at the AAA ATPase site and the proximity-induced reactivity-based inhibition. Together, our findings suggest a chemical genetics approach to decipher AAA protein functions across essential cellular processes and to test hypotheses for developing therapeutics.

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase.

The superfamily 1 helicase nonstructural protein 13 (nsp13) is required for SARS-CoV-2 replication. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the RNA substrate. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be activated >50-fold by piconewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, such as hepatitis C virus NS3, and instead draws stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

Identification and Characterization of a B-Raf Kinase α-Helix Critical for the Activity of MEK Kinase in MAPK Signaling.

In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ∼3% of all cancers, and many drugs target the ATP binding site of the enzyme for its inhibition. Because B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an α-helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We assessed the binding between B-Raf mutants and MEK using pull downs and biolayer interferometry and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this α-helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf α-helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas.

Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex.

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.

High Levels of Glutaminase II Pathway Enzymes in Normal and Cancerous Prostate Suggest a Role in 'Glutamine Addiction'.

Abstract: Many tumors readily convert l-glutamine to α-ketoglutarate. This conversion is almost invariably described as involving deamidation of l-glutamine to l-glutamate followed by a transaminase (or dehydrogenase) reaction. However, mammalian tissues possess another pathway for conversion of l-glutamine to α-ketoglutarate, namely the glutaminase II pathway: l-Glutamine is transaminated to α-ketoglutaramate, which is then deamidated to α-ketoglutarate by ω-amidase. Here we show that glutamine transaminase and ω-amidase specific activities are high in normal rat prostate. Immunohistochemical analyses revealed that glutamine transaminase K (GTK) and ω-amidase are present in normal and cancerous human prostate and that expression of these enzymes increases in parallel with aggressiveness of the cancer cells. Our findings suggest that the glutaminase II pathway is important in providing anaplerotic carbon to the tricarboxylic acid (TCA) cycle, closing the methionine salvage pathway, and in the provision of citrate carbon in normal and cancerous prostate. Finally, our data also suggest that selective inhibitors of GTK and/or ω-amidase may be clinically important for treatment of prostate cancer. In conclusion, the demonstration of a prominent glutaminase II pathway in prostate cancer cells and increased expression of the pathway with increasing aggressiveness of tumor cells provides a new perspective on 'glutamine addiction' in cancers.

Mechanism of BRAF Activation through Biochemical Characterization of the Recombinant Full-Length Protein.

BRAF kinase plays an important role in mitogen-activated protein kinase (MAPK) signaling and harbors activating mutations in about half of melanomas and in a smaller percentage in many other cancers. Despite its importance, few in vitro studies have been performed to characterize the biochemical properties of full-length BRAF. Herein, a strategy to generate an active, intact form of BRAF protein suitable for in vitro enzyme kinetics is described. It is shown that purified, intact BRAF protein autophosphorylates the kinase activation loop and this can be enhanced by binding the MEK protein substrate through an allosteric mechanism. These studies provide in vitro evidence that BRAF selectively binds to active RAS and that the BRAF/CRAF heterodimer is the most active form, relative to their respective homodimers. Full-length BRAF analysis with small-molecule BRAF inhibitors shows that two drugs, dabrafenib and vemurafenib, can modestly enhance kinase activity of BRAF at low concentration. Taken together, this characterization of intact BRAF contributes to a framework for understanding its role in cell signaling.

N-(7-Cyano-6-(4-fluoro-3-(2-(3-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-yl)cyclopropanecarboxamide (TAK632) Promotes Inhibition of BRAF through the Induction of Inhibited Dimers.

BRAFV600E is the most common activating mutation in melanoma and patients treated with BRAFV600E inhibitors all develop resistance within one year. A significant resistance pathway is paradoxical activation (transactivation) involving BRAF dimers, whereby an inhibitor bound protein subunit allosterically activates the other subunit. We recently reported on dimeric BRAFV600E -selective vemurafenib inhibitors that stabilize an inactive αC-out/αC-out homodimeric conformation with improved inhibitor potency and selectivity in vitro. We set out to extend this strategy to target RAF homo- and heterodimers with the pan-RAF inhibitor TAK632 in dimeric configuration. Surprisingly, we find that monomeric TAK632 induces an active αC-in/αC-in BRAF dimer conformation, while dimeric TAK inhibitors cannot promote BRAF dimers and have significantly compromised potency in vitro. These studies uncover the intimate connection between BRAF dimerization and TAK632 mode of inhibition and highlight the importance of understanding the impact of BRAF inhibitors on kinase dimerization.

Case Study - Case Studies in Cystinuria.

The diagnosis and treatment of patients with rare inherited metabolic disorders associated with recurrent and often obstructive kidney stones are important to the prevention of chronic kidney disease or end stage renal disease. Two case studies in this article describe the diagnosis and management of cystinuria, the most common rare kidney stone disorder.

Update of the ICUD-SIU consultation on stone technology behind ureteroscopy.

INTRODUCTION: Ureteroscopy is now the most frequent treatment used around the world for stone disease. Technological advancement, efficiency, safety, and minimally invasiveness of this procedure are some of the reasons for this change of trend. MATERIALS AND METHODS: In this review of the literature, a search of the PubMed database was conducted to identify articles related to ureteroscopy and accessories. The committee assigned by the International Consultation on Urological Disease reviewed all the data and produced a consensus statement relating to the ureteroscopy and all the particularities around this procedure. CONCLUSION: This manuscript provides literatures and recommendations for endourologists to keep them informed in regard to the preoperative, intraoperative, and postoperative consideration in regard of a ureteroscopy.

Chemically Linked Vemurafenib Inhibitors Promote an Inactive BRAFV600E Conformation.

The BRAF kinase, within the mitogen activated protein kinase (MAPK) signaling pathway, harbors activating mutations in about half of melanomas and to a significant extent in many other cancers. A single valine to glutamic acid substitution at residue 600 (BRAFV600E) accounts for about 90% of these activating mutations. While BRAFV600E-selective small molecule inhibitors, such as debrafenib and vemurafenib, have shown therapeutic benefit, almost all patients develop resistance. Resistance often arises through reactivation of the MAPK pathway, typically through mutation of upstream RAS, downstream MEK, or splicing variants. RAF kinases signal as homo- and heterodimers, and another complication associated with small molecule BRAFV600E inhibition is drug-induced allosteric activation of a wild-type RAF subunit (BRAF or CRAF) of the kinase dimer, a process called "transactivation" or "paradoxical activation." Here, we used BRAFV600E and vemurafenib as a model system to develop chemically linked kinase inhibitors to lock RAF dimers in an inactive conformation that cannot undergo transactivation. This structure-based design effort resulted in the development of Vem-BisAmide-2, a compound containing two vemurafenib molecules connected by a bis amide linker. We show that Vem-BisAmide-2 has comparable inhibitory potency as vemurafenib to BRAFV600E both in vitro and in cells but promotes an inactive dimeric BRAFV600E conformation unable to undergo transactivation. The crystal structure of a BRAFV600E/Vem-BisAmide-2 complex and associated biochemical studies reveal the molecular basis for how Vem-BisAmide-2 mediates selectivity for an inactive over an active dimeric BRAFV600E conformation. These studies have implications for targeting BRAFV600E/RAF heterodimers and other kinase dimers for therapy.

HIV Serodisclosure and Sexual Behavior During International Travel.

When traveling internationally, HIV serodisclosure and knowledge of partners' serostatus were hampered by the lack of a common language. Condomless anal intercourse was less likely to occur in partnerships where HIV serostatus was not disclosed or known. Taken together, these observations suggest that language barriers may affect sexual decision making.

The Post-Ureteroscopic Lesion Scale (PULS): a multicenter video-based evaluation of inter-rater reliability.

PURPOSE: The Post-Ureteroscopic Lesion Scale (PULS) offers a simple grading system for the description of ureteral lesions after ureteroscopy. In this article, we present the results of a video-based multicenter evaluation of the inter-rater reliability of clinically important PULS grades 0-3. METHODS: Video sequences at the end of ureteroscopy (final passage) were recorded for 100 consecutive patients at a single institution and assessed by experienced urologists (n = 20) and senior residents (n = 17) at 19 international centers. The cohort included only patients with lesions grades 0-3 (with grades 2 and 3 subsumed as 2 + since distinction is defined by an extravasation of contrast medium in fluoroscopy). The gradings were evaluated for inter-rater reliability and in terms of simplicity, validity, comprehensibility, reproducibility, and usefulness. RESULTS: Overall, inter-rater reliability was high (Kendall's W = 0.69, p < 0.001) and was comparable between specialists (Kendall's W = 0.69, p < 0.001) and residents (Kendall's W = 0.71, p < 0.001). The matched ratings showed grade 0 in 43.0 % of patients and grades 1 or 2 + in 44.0 and 13.0 % of patients, respectively. Results of the questionnaires indicated a high degree of acceptance, with an overall rating of 1.76 (1.64-1.93 for different items, scale 1-6). CONCLUSIONS: Inter-rater reliability of the endoscopically assessable PULS was high among urologists with different levels of experience in different countries worldwide. The validated PULS system may be used for standardized reporting of ureteral lesions/injuries after ureteroscopy. In addition, PULS will enable more selective standardization of indications for postoperative DJ stenting based on the randomized controlled trials.

Health-related quality of life (HRQoL) in cystine compared with non-cystine stone formers.

Cystinuria is a genetic cause of recurrent kidney stones which may be more recurrent and larger than more common non-cystine stones. They may have a greater impact on health-related quality of life (HRQoL). We measured this impact by surveying HRQoL in patients with stones, comparing non-cystine stone formers (NCSF) to cystine stone formers (CYSF) and both groups to normative values of the US population. We used SF-36v2 via an internet instrument. CYSF patients were recruited via cystinuria-related websites, two patient advocacy groups, and an active endourology practice. NCSF patients were recruited from the same practice and by email. Total n surveyed with scorable data: 214 CYSF and 81 NCSF. The participants included 128 men and 161 women. The group of CYSF were significantly younger (39 vs. 54 years) and suffered longer from kidney stones (255 vs. 136 months). CYSF patients had significantly more episodes of stones than NCSF patients in the last year (N = 108 CYSF, N = 20 NCSF). More frequent stones in the last year and mental comorbidities most often predicted worse scores in the individual HRQoL domains. However, cystine stone composition was a significant predictor of worse scores only for role emotional. Better scores in all SF-36 domains were associated with greater time since the last kidney stone event. Although kidney stones are often transient, kidney stone formers, regardless of stone composition, have a worse HRQoL than the standard US population, which has a normative score of 50, such as general health (41.2 ± 12.8), bodily pain (46.5 ± 11.8) and mental health (45.1 ± 12.6). CYSF are more frequent and severe stone formers compared with NCSF with a resulting greater, direct impact on the HRQoL of CYSF patients. Whether preventive strategies for cystinuria are being properly utilized by practitioners, and which strategies are most effective, should be established.

3.0 T multiparametric prostate MRI using pelvic phased-array coil: utility for tumor detection prior to biopsy.

OBJECTIVE: To evaluate the role of multiparametric magnetic resonance imaging (MRI) performed in men without a biopsy-proven diagnosis of prostate cancer using follow-up biopsy as the reference standard. MATERIALS AND METHODS: Forty-two patients without biopsy-proven cancer and who underwent MRI were included. In all patients, MRI was performed at 3T using a pelvic phased-array coil and included T2-weighted imaging, diffusion-weighted imaging, and dynamic contrast-enhanced imaging. Thirteen had undergone no previous biopsy, and 29 had undergone at least 1 previous negative biopsy. All patients underwent prostate biopsy following MRI. Two fellowship-trained radiologists in consensus reviewed all cases and categorized each lobe as positive or negative for tumor. These interpretations were correlated with findings on post-MRI biopsy. RESULTS: Follow-up biopsy was positive in 23 lobes in 15 patients (36% of study cohort). On a per-patient basis, MRI had a sensitivity of 100%, specificity of 74%, positive predictive value (PPV) of 68%, and negative predictive value (NPV) of 100%. On a per-lobe basis, MRI had a sensitivity of 65%, specificity of 84%, PPV of 60%, and NPV of 86%. There was a nearly significant association between Gleason score and tumor detection on MRI (P = 0.072). CONCLUSIONS: In our sample, MRI had 100% sensitivity in predicting the presence of tumor on subsequent biopsy on a per-patient basis, suggesting a possible role for MRI in selecting patients with an elevated prostatic specific antigen (PSA) to undergo prostate biopsy. However, MRI had weaker specificity for prediction of a subsequent positive biopsy, as well as weaker sensitivity for tumor on a per-lobe basis, indicating that in patients with a positive MRI result, tissue sampling remains necessary for confirmation of the diagnosis as well as for treatment planning.