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Everolimus (EVE) combined with letrozole is an approved treatment for hormone receptor‐positive/human epidermal growth factor receptor 2‐negative (HR+/HER2−) advanced breast cancer (ABC). However, predictive biomarkers for EVE efficacy remain undefined. In the phase 2 MIRACLE trial, we performed digital spatial profiling (DSP) on pretreatment tumor samples from 21 patients receiving EVE plus letrozole. Patients were divided into resistant and sensitive groups based on their best response to EVE. A total of 119 regions across three compartments—tumor, leukocytes, and stroma—were profiled for immune and transcriptomic markers. Responders had significantly higher fibroblast infiltration in PANCK+ (p = 0.011) and CD45−/PANCK− (p = 0.043) regions, whereas non‐responders exhibited increased neutrophils in CD45+ (p = 0.0061) and PANCK+ (p = 0.03) regions. Prolactin‐induced protein (PIP) mRNA expression was significantly elevated in non‐responders in both PANCK+ (p < 0.0001) and CD45−/PANCK− (p = 0.0006) regions. PIP mRNA expression was found to be associated with EVE resistance and unfavorable progression‐free survival (PFS). PIP mRNA expression and specific immune‐stromal features are associated with resistance to EVE. These findings suggest the potential of PIP as a spatially resolved predictive biomarker for patient stratification in HR+/HER2− ABC. In the phase 2 MIRACLE trial, we performed digital spatial profiling (DSP) on pretreatment tumor samples from 21 patients receiving EVE plus letrozole. Patients were divided into resistant and sensitive groups based on their best response to EVE. Responders had significantly higher fibroblast infiltration in PANCK+ and CD45−/PANCK− regions, whereas non‐responders exhibited increased neutrophils in CD45+ and PANCK+ regions. Prolactin‐induced protein (PIP) mRNA expression was found to be associated with EVE resistance and unfavorable progression‐free survival (PFS).

Pathogenic variants in KCNQ2 encoding for Kv7.2 potassium channel subunits have been found in patients affected by widely diverging epileptic phenotypes, ranging from Self-Limiting Familial Neonatal Epilepsy (SLFNE) to severe Developmental and Epileptic Encephalopathy (DEE). Thus, understanding the pathogenic molecular mechanisms of KCNQ2 variants and their correlation with clinical phenotypes has a relevant impact on the clinical management of these patients. In the present study, the genetic, biochemical, and functional effects prompted by two variants, each found in a non-familial SLNE or a DEE patient but both affecting nucleotides at the KCNQ2 intron 6-exon 7 boundary, have been investigated to test whether and how they affected the splicing process and to clarify whether such mechanism might play a pathogenetic role in these patients. Analysis of KCNQ2 mRNA splicing in patient-derived lymphoblasts revealed that the SLNE-causing intronic variant (c.928-1G > C) impeded the use of the natural splice site, but lead to a 10-aa Kv7.2 in frame deletion (Kv7.2 p.G310Δ10); by contrast, the DEE-causing exonic variant (c.928G > A) only had subtle effects on the splicing process at this site, thus leading to the synthesis of a full-length subunit carrying the G310S missense variant (Kv7.2 p.G310S). Patch-clamp recordings in transiently-transfected CHO cells and primary neurons revealed that both variants fully impeded Kv7.2 channel function, and exerted strong dominant-negative effects when co-expressed with Kv7.2 and/or Kv7.3 subunits. Notably, Kv7.2 p.G310S, but not Kv7.2 p.G310Δ10, currents were recovered upon overexpression of the PIP 2 -synthesizing enzyme PIP5K, and/or CaM; moreover, currents from heteromeric Kv7.2/Kv7.3 channels incorporating either Kv7.2 mutant subunits were differentially regulated by changes in PIP 2 availability, with Kv7.2/Kv7.2 G310S/Kv7.3 currents showing a greater sensitivity to PIP 2 depletion when compared to those from Kv7.2/Kv7.2 G310Δ10/Kv7.3 channels. Altogether, these results suggest that the two variants investigated differentially affected the splicing process at the intron 6-exon 7 boundary, and led to the synthesis of Kv7.2 subunits showing a differential sensitivity to PIP 2 and CaM regulation; more studies are needed to clarify how such different functional properties contribute to the widely-divergent clinical phenotypes.

Water movement across root tissues occurs by parallel apoplastic, symplastic, and transcellular pathways that the plant can control to a certain extent. Because water channels or aquaporins (AQPs) play an important role in regulating water flow, studies on AQP mRNA and protein expression in different root tissues are essential. Here, we quantified and localized the expression of Zea mays plasma membrane AQPs (ZmPIPs) in primary root tip using in situ and quantitative RT-PCR and immunodetection approaches. All ZmPIP genes except ZmPIP2;7 were expressed in primary roots. Expression was found to be dependent on the developmental stage of the root, with, in general, an increase in expression towards the elongation and mature zones. Two genes, ZmPIP1;5 and ZmPIP2;5, showed the greatest increase in expression (up to 11- and 17-fold, respectively) in the mature zone, where they accounted for 50% of the total expressed ZmPIPs. The immunocytochemical localization of ZmPIP2;1 and ZmPIP2;5 in the exodermis and endodermis indicated that they are involved in root radial water movement. In addition, we detected a polar localization of ZmPIP2;5 to the external periclinal side of epidermal cells in root apices, suggesting an important role in water uptake from the root surface. Finally, protoplast swelling assays showed that root cells display a variable, but globally low, osmotic water permeability coefficient (Pf < 10 microm/s). However, the presence of a population of cells with a higher Pf (up to 26 microm/s) in mature zone of the root might be correlated with the increased expression of several ZmPIP genes.