Comparison of dose selection based on target engagement versus inhibition of receptor-ligand interaction for checkpoint inhibitors.

Immune checkpoint inhibitors block the interaction between a receptor on one cell and its ligand on another cell, thus preventing the transduction of an immunosuppressive signal. While inhibition of the receptor-ligand interaction is key to the pharmacological activity of these drugs, it can be technically challenging to measure these intercellular interactions directly. Instead, target engagement (or receptor occupancy) is commonly measured, but may not always be an accurate predictor of receptor-ligand inhibition, and can be misleading when used to inform clinical dose projections for this class of drugs. In this study, a mathematical model explicitly representing the intercellular receptor-ligand interaction is used to compare dose prediction based on target engagement or receptor-ligand inhibition for two checkpoint inhibitors, atezolizumab and magrolimab. For atezolizumab, there is little difference between target engagement and receptor-ligand inhibition, but for magrolimab, the model predicts that receptor-ligand inhibition is significantly less than target engagement. The key variables explaining the difference between these two drugs are the relative concentrations of the target receptors and their ligands. Drug-target affinity and receptor-ligand affinity can also have divergent effects on target engagement and inhibition. These results suggest that it is important to consider ligand-receptor inhibition in addition to target engagement and demonstrate the impact of using modeling for efficacious dose estimation.

Excited-State Identification of a Nickel-Bipyridine Photocatalyst by Time-Resolved X-ray Absorption Spectroscopy.

Photoassisted catalysis using Ni complexes is an emerging field for cross-coupling reactions in organic synthesis. However, the mechanism by which light enables and enhances the reactivity of these complexes often remains elusive. Although optical techniques have been widely used to study the ground and excited states of photocatalysts, they lack the specificity to interrogate the electronic and structural changes at specific atoms. Herein, we report metal-specific studies using transient Ni L- and K-edge X-ray absorption spectroscopy of a prototypical Ni photocatalyst, (dtbbpy)Ni(o-tol)Cl (dtb = 4,4'-di-tert-butyl, bpy = bipyridine, o-tol = ortho-tolyl), in solution. We unambiguously confirm via direct experimental evidence that the long-lived (∼5 ns) excited state is a tetrahedral metal-centered triplet state. These results demonstrate the power of ultrafast X-ray spectroscopies to unambiguously elucidate the nature of excited states in important transition-metal-based photocatalytic systems.

Molecular basis for plasma membrane recruitment of PI4KA by EFR3.

The lipid kinase phosphatidylinositol 4 kinase III alpha (PI4KIIIα/PI4KA) is a master regulator of the lipid composition and asymmetry of the plasma membrane. PI4KA exists primarily in a heterotrimeric complex with its regulatory proteins TTC7 and FAM126. Fundamental to PI4KA activity is its targeted recruitment to the plasma membrane by the lipidated proteins EFR3A and EFR3B. Here, we report a cryo-EM structure of the C-terminus of EFR3A bound to the PI4KA-TTC7B-FAM126A complex, with extensive validation using both hydrogen deuterium exchange mass spectrometry (HDX-MS), and mutational analysis. The EFR3A C-terminus undergoes a disorder-order transition upon binding to the PI4KA complex, with an unexpected direct interaction with both TTC7B and FAM126A. Complex disrupting mutations in TTC7B, FAM126A, and EFR3 decrease PI4KA recruitment to the plasma membrane. Multiple post-translational modifications and disease linked mutations map to this site, providing insight into how PI4KA membrane recruitment can be regulated and disrupted in human disease.

Spatial transcriptomic analysis reveals local effects of intratumoral fusobacterial infection on DNA damage and immune signaling in rectal cancer.

Mucinous colorectal cancer (CRC) is a common histological subtype of colorectal adenocarcinoma, associated with a poor response to chemoradiotherapy. The commensal facultative anaerobes fusobacteria, have been associated with poor prognosis specifically in mesenchymal CRC. Interestingly, fusobacterial infection is especially prevalent in mucinous CRC. The objective of this study was therefore to increase our understanding of beneficial and detrimental effects of fusobacterial infection, by contrasting host cell signaling and immune responses in areas of high vs. low infection, using mucinous rectal cancer as a clinically relevant example. We employed spatial transcriptomic profiling of 106 regions of interest from 8 mucinous rectal cancer samples to study gene expression in the epithelial and immune segments across regions of high versus low fusobacterial infection. Fusobacteria high regions were associated with increased oxidative stress, DNA damage, and P53 signaling. Meanwhile regions of low fusobacterial prevalence were characterized by elevated JAK-STAT, Il-17, Il-1, chemokine and TNF signaling. Immune masks within fusobacterial high regions were characterized by elevated proportions of cytotoxic (CD8+) T cells (p = 0.037), natural killer (NK) cells (p < 0.001), B-cells (p < 0.001), and gamma delta T cells (p = 0.003). Meanwhile, fusobacteria low regions were associated with significantly greater M2 macrophage (p < 0.001), fibroblast (p < 0.001), pericyte (p = 0.002), and endothelial (p < 0.001) counts.

Transanal ileal pouch-anal anastomosis: A systematic review and meta-analysis of technical approaches and clinical outcomes.

PURPOSE: Transanal minimally invasive surgery has theoretical advantages for ileal pouch-anal anastomosis surgery. We performed a systematic review assessing technical approaches to transanal IPAA (Ta-IPAA) and meta-analysis comparing outcomes to transabdominal (abd-IPAA) approaches. METHODS: Three databases were searched for articles investigating Ta-IPAA outcomes. Primary outcome was anastomotic leak rate. Secondary outcomes included conversion rate, post operative morbidity, and length of stay (LoS). Staging, plane of dissection, anastomosis, extraction site, operative time, and functional outcomes were also assessed. RESULTS: Searches identified 13 studies with 404 unique Ta-IPAA and 563 abd-IPAA patients. Anastomotic leak rates were 6.3% and 8.4% (RD 0, 95% CI -0.066 to 0.065, p = 0.989) and conversion rates 2.5% and 12.5% (RD -0.106, 95% CI -0.155 to -0.057, p = 0.104) for Ta-IPAA and abd-IPAA. Average LoS was one day shorter (MD -1, 95% CI -1.876 to 0.302, p = 0.007). A three-stage approach was most common (47.6%), operative time was 261(± 60) mins, and total mesorectal excision and close rectal dissection were equally used (49.5% vs 50.5%). Functional outcomes were similar. Lack of randomised control trials, case-matched series, and significant study heterogeneity limited analysis, resulting in low to very low certainty of evidence. CONCLUSIONS: Analysis demonstrated the feasibility and safety of Ta-IPAA with reduced LoS, trend towards less conversions, and comparable anastomotic leak rates and post operative morbidity. Though results are encouraging, they need to be interpreted with heterogeneity and selection bias in mind. Robust randomised clinical trials are warranted to adequately compare ta-IPAA to transabdominal approaches.

ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6-specific nanobody restricts M. tuberculosis growth in macrophages.

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH-dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

The effect of implementing a transanal minimally invasive surgical programme for the local excision of early rectal neoplasia on outcomes in a tertiary referral rectal cancer centre.

Transanal minimally invasive surgery (TAMIS) is a surgical alternative to proctectomy in the management of complex rectal polyps and early rectal cancers. In 2016, our institution introduced a TAMIS programme. The purpose of this study was to evaluate changes in practice and outcomes in our institution in the 3 years before and after the implementation of TAMIS. We conducted a retrospective analysis of a prospective database of patients who underwent proctectomy or TAMIS for the management of complex rectal polyps or early rectal cancers at our institution between 2013 and 2018. 96 patients were included in this study (41 proctectomy vs 55 TAMIS). A significant reduction was noted in the number of proctectomies performed in the 3 years after the implementation of TAMIS as compared to the 3 years before (13 vs 28) ( P  < 0.001); 43% of patients ( n  = 12) who underwent proctectomy in the period prior to implementation of TAMIS were American Society of Anaesthesiologists grade III, as compared to only 15% ( n  = 2) of patients during the period following TAMIS implementation ( P  = 0.02). TAMIS was associated with a significant reduction in length of inpatient stay ( P  < 0.001). Oncological outcomes were comparable between groups (log rank P  = 0.83). Our findings support TAMIS as a safe and effective alternative to radical resection. The availability of TAMIS has resulted in a significant reduction in the number of comorbid patients undergoing proctectomy at our institution. Consequently, we have observed a significant reduction in postoperative complications over this time period.

Overall Survival with Adjuvant Pembrolizumab in Renal-Cell Carcinoma.

BACKGROUND: Adjuvant pembrolizumab therapy after surgery for renal-cell carcinoma was approved on the basis of a significant improvement in disease-free survival in the KEYNOTE-564 trial. Whether the results regarding overall survival from the third prespecified interim analysis of the trial would also favor pembrolizumab was uncertain. METHODS: In this phase 3, double-blind, placebo-controlled trial, we randomly assigned (in a 1:1 ratio) participants with clear-cell renal-cell carcinoma who had an increased risk of recurrence after surgery to receive pembrolizumab (at a dose of 200 mg) or placebo every 3 weeks for up to 17 cycles (approximately 1 year) or until recurrence, the occurrence of unacceptable toxic effects, or withdrawal of consent. A significant improvement in disease-free survival according to investigator assessment (the primary end point) was shown previously. Overall survival was the key secondary end point. Safety was a secondary end point. RESULTS: A total of 496 participants were assigned to receive pembrolizumab and 498 to receive placebo. As of September 15, 2023, the median follow-up was 57.2 months. The disease-free survival benefit was consistent with that in previous analyses (hazard ratio for recurrence or death, 0.72; 95% confidence interval [CI], 0.59 to 0.87). A significant improvement in overall survival was observed with pembrolizumab as compared with placebo (hazard ratio for death, 0.62; 95% CI, 0.44 to 0.87; P = 0.005). The estimated overall survival at 48 months was 91.2% in the pembrolizumab group, as compared with 86.0% in the placebo group; the benefit was consistent across key subgroups. Pembrolizumab was associated with a higher incidence of serious adverse events of any cause (20.7%, vs. 11.5% with placebo) and of grade 3 or 4 adverse events related to pembrolizumab or placebo (18.6% vs. 1.2%). No deaths were attributed to pembrolizumab therapy. CONCLUSIONS: Adjuvant pembrolizumab was associated with a significant and clinically meaningful improvement in overall survival, as compared with placebo, among participants with clear-cell renal-cell carcinoma at increased risk for recurrence after surgery. (Funded by Merck Sharp and Dohme, a subsidiary of Merck; KEYNOTE-564 ClinicalTrials.gov number, NCT03142334.).

Meta-analysis of SATB2 immunohistochemical expression in colorectal cancer versus primary ovarian mucinous neoplasms.

BACKGROUND: Reliably distinguishing primary ovarian mucinous neoplasms (POMNs) from metastatic colorectal cancers (CRCs) is both challenging to the histopathologist and of great clinical importance. Special AT-rich sequence binding protein-2 (SATB2) has emerged as a useful diagnostic immunohistochemical marker of colorectal cancer. This meta-analysis compares SATB2 expression in POMNs and CRC. METHODS: A systematic literature search for relevant studies was conducted. Meta-analysis of SATB2 positivity was undertaken using a random effects model. RESULTS: Seven studies including 711 CRCs and 528 POMNs were included. SATB2 positivity was seen in 81 % (95 % CI: 72-88 %) of CRCs and 4 % (95 % CI: 1-11 %) of POMNs. Variation was seen in immunohistochemical methods used for SATB2 detection and threshold for positivity. CONCLUSION: SATB2 staining remains high in CRC and low in POMNs, supporting its use in differentiating these two pathologies with vastly differing prognosis and treatment.

Validation of SeptiCyte RAPID to Discriminate Sepsis from Non-Infectious Systemic Inflammation.

(1) Background: SeptiCyte RAPID is a molecular test for discriminating sepsis from non-infectious systemic inflammation, and for estimating sepsis probabilities. The objective of this study was the clinical validation of SeptiCyte RAPID, based on testing retrospectively banked and prospectively collected patient samples. (2) Methods: The cartridge-based SeptiCyte RAPID test accepts a PAXgene blood RNA sample and provides sample-to-answer processing in ~1 h. The test output (SeptiScore, range 0-15) falls into four interpretation bands, with higher scores indicating higher probabilities of sepsis. Retrospective (N = 356) and prospective (N = 63) samples were tested from adult patients in ICU who either had the systemic inflammatory response syndrome (SIRS), or were suspected of having/diagnosed with sepsis. Patients were clinically evaluated by a panel of three expert physicians blinded to the SeptiCyte test results. Results were interpreted under either the Sepsis-2 or Sepsis-3 framework. (3) Results: Under the Sepsis-2 framework, SeptiCyte RAPID performance for the combined retrospective and prospective cohorts had Areas Under the ROC Curve (AUCs) ranging from 0.82 to 0.85, a negative predictive value of 0.91 (sensitivity 0.94) for SeptiScore Band 1 (score range 0.1-5.0; lowest risk of sepsis), and a positive predictive value of 0.81 (specificity 0.90) for SeptiScore Band 4 (score range 7.4-15; highest risk of sepsis). Performance estimates for the prospective cohort ranged from AUC 0.86-0.95. For physician-adjudicated sepsis cases that were blood culture (+) or blood, urine culture (+)(+), 43/48 (90%) of SeptiCyte scores fell in Bands 3 or 4. In multivariable analysis with up to 14 additional clinical variables, SeptiScore was the most important variable for sepsis diagnosis. A comparable performance was obtained for the majority of patients reanalyzed under the Sepsis-3 definition, although a subgroup of 16 patients was identified that was called septic under Sepsis-2 but not under Sepsis-3. (4) Conclusions: This study validates SeptiCyte RAPID for estimating sepsis probability, under both the Sepsis-2 and Sepsis-3 frameworks, for hospitalized patients on their first day of ICU admission.

Dialysis disequilibrium syndrome in neurosurgery: literature review and illustrative case example.

INTRODUCTION: The dialysis disequilibrium syndrome (DDS) is a complication in those undergoing dialysis for chronic kidney disease (CKD) or acute kidney injury (AKI), characterized by nonspecific symptoms that may progress to coma and death secondary to cerebral edema. This syndrome is associated with rapid change in electrolytes during dialysis with changes in intracranial pressure (ICP) and may have a higher incidence in the elderly neurosurgical patient population. METHODS: Literature review and illustrative case example. RESULTS: A 62-year-old female presented with acute mental status change during hemodialysis (HD), with a history of a nonsurgical acute subdural hematoma (SDH) 10 days prior. Imaging showed a conversion of the acute SDH to chronic SDH of 12.2 mm in size with a 14.1 midline shift, for which she underwent a hemicraniectomy with SDH evacuation, with a gradual return to baseline. The literature review identified 5 publications meeting the inclusion criteria. Major theories of DDS include a reverse urea effect, intracerebral acidosis, idiogenic osmoles, and local inflammation. This complication may occur more frequently in the elderly neurosurgical patient population, likely due to age-related comorbidities, preexisting neurological insult, and increased permeability of the blood-brain barrier (BBB), leading to cerebral edema. CONCLUSION: DDS is a rare and potentially fatal complication of HD that may have a higher incidence in the elderly neurosurgical patient population, yet remains to be fully understood. Further study is recommended to characterize the pathophysiological mechanism and incidence of DDS in neurosurgical patients.

Contemporary perioperative outcomes after total abdominal colectomy for ulcerative colitis in a tertiary referral centre.

OBJECTIVE: Colectomy for ulcerative colitis (UC) is common despite therapeutic advances. Post-operative morbidity and mortality demonstrate an association between hospital volumes and outcomes. This single-centre retrospective study examines outcomes after emergency colectomy for UC. METHODS: Patient demographics, perioperative variables and outcomes were collected in Beaumont Hospital between 2010 and 2023. Univariant analysis was used to assess relationships between perioperative variables and morbidity and length of stay (LOS). RESULTS: A total of 115 patients underwent total abdominal colectomy with end ileostomy for UC, 8.7 (±3.8) per annum. Indications were refractory acute severe colitis (88.7%), toxic megacolon (6.1%), perforation (4.3%), or obstruction (0.9%). Over 80% of cases were performed laparoscopically. Pre-operative steroid (93%) and biologic (77.4%) use was common. Median post-operative LOS was 8 days (interquartile range 6-12). There were no 30-day mortalities, and 30-day post-operative morbidity was 38.3%. There was no association between time to colectomy ( P  = 0.85) or biologic use ( P  = 0.24) and morbidity. Increasing age was associated with prolonged LOS ( P  = 0.01). Laparoscopic approach (7 vs. 12 days P  =0.01, 36.8% vs. 45% P  = 0.66) was associated with reduced LOS and morbidity. CONCLUSION: This study highlights contemporary outcomes after emergency colectomy for UC at a specialist high-volume, tertiary referral centre, and superior outcomes after laparoscopic surgery in the biologic era.

Acute Macrocystic Thoracic Schwannoma: Systematic Review and Illustrative Case Example.

BACKGROUND: Schwannomas are benign peripheral nerve sheath tumors arising from myelinating Schwann cells. Although macrocystic changes are regularly encountered in schwannoma variants such as vestibular nerve tumors, they are exceedingly rare among spinal neoplasms. METHODS: Case report and systematic review of 4 databases (Ovid Medline, PubMed, Science Direct, and SCOPUS) from inception to present. All peer-reviewed publications reporting intradural cystic thoracic schwannoma were included. RESULTS: We identified 8 publications documenting 9 cases of cystic thoracic schwannoma. Four were female, 5 male; median age was 41 years (range, 27-80). Presentations ranged from incidental to pain, sensory changes, lower extremity paresis, or bowel/bladder dysfunction. Characteristic radiographic findings included T1 hypointensity, T2 hyperintensity, and cord effacement or compression. The present case followed a similar pattern: a 52-year-old male presented with worsening bilateral lower extremity weakness, low back pain, and gait dysfunction, worsening over 3 days. Examination also revealed decreased left lower extremity sensation. Imaging identified a well-delineated intradural, extramedullary macrocystic extending over T7-T10. The patient underwent a laminectomy resulting in complete tumor resection and restoration of intact neurologic function. Final pathology confirmed benign cystic schwannoma. CONCLUSIONS: Macrocystic thoracic schwannomas are exceedingly rare and lack a comprehensive scheme for clinical classification of their natural history and pathogenesis. We report the 10th case of such a schwannoma, and the first associated systematic review. Although macrocystic thoracic schwannomas are not frequently encountered, accurate diagnosis and appropriate neurosurgical treatment is critical in these vulnerable patients, given the opportunity for excellent functional outcomes following neurosurgical treatment.

Leiomyogenic Tumor of the Spine: A Systematic Review.

The study cohort consisted of 83 patients with a mean age of 49.55 (SD 13.72) with a female preponderance (60 patients). Here, 32.14% of patients had primary LTS; the remaining were metastases. Clinical presentation included nonspecific back pain (57.83%), weakness (21.69%) and radicular pain (18.07%). History of uterine neoplasia was found in 33.73% of patients. LTS preferentially affected the thoracic spine (51.81%), followed by the lumbar (21.67%) spine. MRI alone was the most common imaging modality (33.33%); in other cases, it was used with CT (22.92%) or X-ray (16.67%); 19.23% of patients had Resection/Fixation, 15.38% had Total en bloc spondylectomy, and 10.26% had Corpectomy. A minority of patients had laminectomy and decompression. Among those with resection, 45.83% had a gross total resection, 29.17% had a subtotal resection, and 16.67% had a near total resection. Immunohistochemistry demonstrated positivity for actin (43.37%), desmin (31.33%), and Ki67 (25.30). At a follow-up of 19.3 months, 61.97% of patients were alive; 26.25% of 80 patients received no additional treatment, 23.75% received combination radiotherapy and chemotherapy, only chemotherapy was given to 20%, and radiotherapy was given to 17.5%. Few (2.5%) had further resection. For an average of 12.50 months, 42.31% had no symptoms, while others had residual (19.23%), other metastasis (15.38%), and pain (7.69%). On follow-up of 29 patients, most (68.97%) had resolved symptoms; 61.97% of the 71 patients followed were alive.

Spartin-mediated lipid transfer facilitates lipid droplet turnover.

Lipid droplets (LDs) are organelles critical for energy storage and membrane lipid homeostasis, whose number and size are carefully regulated in response to cellular conditions. The molecular mechanisms underlying lipid droplet biogenesis and degradation, however, are not well understood. The Troyer syndrome protein spartin (SPG20) supports LD delivery to autophagosomes for turnover via lipophagy. Here, we characterize spartin as a lipid transfer protein whose transfer ability is required for LD degradation. Spartin copurifies with phospholipids and neutral lipids from cells and transfers phospholipids in vitro via its senescence domain. A senescence domain truncation that impairs lipid transfer in vitro also impairs LD turnover in cells while not affecting spartin association with either LDs or autophagosomes, supporting that spartin's lipid transfer ability is physiologically relevant. Our data indicate a role for spartin-mediated lipid transfer in LD turnover.

Transanal minimally invasive surgery (TAMIS) for local excision of benign and malignant rectal neoplasia: a 7-year experience.

PURPOSE: Transanal minimally invasive surgery (TAMIS) is an advanced transanal platform that can be utilised to perform high-quality local excision (LE) of rectal neoplasia. This study describes clinical and midterm oncological outcomes from a single unit's 7-year experience with TAMIS. METHODS: Consecutive patients who underwent TAMIS LE at our institution between January 1st, 2016, and December 31st, 2022, were identified from a prospectively maintained database. Indication for TAMIS LE was benign lesions not amenable to endoscopic excision or histologically favourable early rectal cancers. The primary endpoints were resection quality, disease recurrence and peri-operative outcomes. The Kaplan-Meier survival analyses were used to describe disease-free survival (DFS) for patients with rectal adenocarcinoma that did not receive immediate salvage proctectomy. RESULTS: There were 168 elective TAMIS LE procedures performed for 102 benign and 66 malignant lesions. Overall, a 95.2% negative margin rate was observed, and 96.4% of lesions were submitted without fragmentation. Post-operative morbidity was recorded in 8.3% of patients, with post-operative haemorrhage, being the most common complication encountered. The mean follow-up was 17 months (SD 15). Local recurrence occurred in 1.6%, and distant organ metastasis was noted in 1.6% of patients. CONCLUSIONS: For carefully selected patients, TAMIS for local excision of early rectal neoplasia is a valid option with low morbidity that maintains the advantages of organ preservation.

Genomic landscape of patients in a phase II study of zanubrutinib in ibrutinib- and/or acalabrutinib-intolerant patients with B-cell malignancies.

Not available.

The genomic basis of nitrogen utilization efficiency and trait plasticity to improve nutrient stress tolerance in cultivated sunflower.

Maintaining crop productivity is challenging as population growth, climate change, and increasing fertilizer costs necessitate expanding crop production to poorer lands whilst reducing inputs. Enhancing crops' nutrient use efficiency is thus an important goal, but requires a better understanding of related traits and their genetic basis. We investigated variation in low nutrient stress tolerance in a diverse panel of cultivated sunflower genotypes grown under high and low nutrient conditions, assessing relative growth rate (RGR) as performance. We assessed variation in traits related to nitrogen utilization efficiency (NUtE), mass allocation, and leaf elemental content. Across genotypes, nutrient limitation generally reduced RGR. Moreover, there was a negative correlation between vigor (RGR in control) and decline in RGR in response to stress. Given this trade-off, we focused on nutrient stress tolerance independent of vigor. This tolerance metric correlated with the change in NUtE, plasticity for a suite of morphological traits, and leaf element content. Genome-wide associations revealed regions associated with variation and plasticity in multiple traits, including two regions with seemingly additive effects on NUtE change. Our results demonstrate potential avenues for improving sunflower nutrient stress tolerance independent of vigor, and highlight specific traits and genomic regions that could play a role in enhancing tolerance.

ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6 specific nanobody restricts M. tuberculosis growth in macrophages.

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.