Protein functional effector sncRNAs (pfeRNAs) are approximately 30–60 nucleotides (nt), of which the extraction method from plasma has not yet been reported. Silver staining in a high-resolution polyacrylamide gel suggested that the majority of plasma sncRNAs extracted by some broadly used commercial kits were sncRNAs from 100 nt upwards. Additionally, TRIzol’s protocol is for long RNA but not sncRNA recovery. Here, we report a TRIzol-based frozen precipitation method (TFP method), which shows rigor and reproducibility in high yield and quality for plasma sncRNAs approximately 30–60 nt. In contrast to the yields by the commercial kit, plasma sncRNAs extracted by the TFP method enriched more sncRNAs. We used four different pfeRNAs of 34 nt, 45 nt, 53 nt, and 58 nt to represent typical sizes of sncRNAs from 30 to 60 nt and compared their levels in the recovered sncRNAs by the TFP method and by the commercial kit. The TFP method showed lower cycle threshold (CT) values by 2.01–9.17 cycles in 38 plasma samples from 38 patients, including Caucasian, Asian, African American, Latin, Mexican, and those who were a mix of more than one race. In addition, pfeRNAs extracted by two organic-based extraction methods and four commercial kits were undetermined in 22 of 38 samples. Thus, the quick and unbiased TFP method enriches plasma sncRNA ranging from 30 to 60 nt.

The ability to differentiate between benign, suspicious, and malignant pulmonary nodules is imperative for definitive intervention in patients with early stage lung cancers. Here, we report that plasma protein functional effector sncRNAs (pfeRNAs) serve as non-invasive biomarkers for determining both the existence and the nature of pulmonary nodules in a three-stage study that included the healthy group, patients with benign pulmonary nodules, patients with suspicious nodules, and patients with malignant nodules. Following the standards required for a clinical laboratory improvement amendments (CLIA)-compliant laboratory-developed test (LDT), we identified a pfeRNA classifier containing 8 pfeRNAs in 108 biospecimens from 60 patients by sncRNA deep sequencing, deduced prediction rules using a separate training cohort of 198 plasma specimens, and then applied the prediction rules to another 230 plasma specimens in an independent validation cohort. The pfeRNA classifier could (1) differentiate patients with or without pulmonary nodules with an average sensitivity and specificity of 96.2% and 97.35% and (2) differentiate malignant versus benign pulmonary nodules with an average sensitivity and specificity of 77.1% and 74.25%. Our biomarkers are cost-effective, non-invasive, sensitive, and specific, and the qPCR-based method provides the possibility for automatic testing of robotic applications.

A new therapeutic avenue combining Durvalumab with cisplatin-pemetrexed (Durva-CP) has delivered a promising outcome for previously untreated patients with unresectable malignant pleural mesothelioma (MPM) in clinical trials. However, the limited patient response to Durva-CP needs predictors to select optimal candidates and monitor the developed resistance. Protein functional effector sncRNA (pfeRNA) reveals a fundamental mechanism underlying the regulation of protein activity. The common mechanisms underlying durvalumab, cisplatin, and pemetrexed indicate that PD-L1 pfeRNAs (PDLpfeRNAs) are key molecules that control the treatment response. We specified PDLpfeRNAs by sncRNA deep sequencing, confirmed their binding to PD-L1 by immunoprecipitation and reverse pull-down assays, and demonstrated their roles in controlling the interaction behaviors of PD1/L1 through quality-controlled drug development assays. Following the standards required for the CLIA-compliant LDT, we measured their expression levels in 60 plasma biospecimens from 30 unresectable MPM patients enrolled in the PrE0505 Phase II multicenter study. Using the Cox proportional hazards model and Kaplan-Meier analyses, we described their significance in predicting the treatment response of unresectable MPM patients administered Durva-CP as first-line therapy. Two PDLpfeRNAs, PDLpfeRNAa and PDLpfeRNAb, were characterized, confirmed to bind to PD-L1, and identified to control the interaction behaviors of PD-1/L1. Their plasma relative expression levels (REL) demonstrated significant prognostic value for both overall survival (p = 0.0019) and progression-free survival (p = 0.019), and the association remained significant after adjusting for histological subtype (HR 2.59, 95 % CI: 1.00-6.70, p = 0.050) and age (HR 1.03, 95 % CI: 0.98-1.07, p = 0.269). Plasma PDLpfeRNAs are predictors of treatment response of unresectable MPM patients treated with Durva-CP as first-line therapy to select optimal candidates and monitor the developed resistance.

Telomeres progressively shorten with each cell division in human somatic cells, but the mechanisms governing telomere length remain incompletely understood. Although telomerase reverse transcriptase (TERT) is central to telomere maintenance, the discrepancy between TERT level and telomere length highlights the unidentified molecules that regulate TERT’s functional activities. Here, we identify novel TERT-associated protein functional effector sncRNAs (TpfeRNAs) that modulate telomerase activity and telomere length during cellular senescence of normal human bronchial epithelial (NHBE) cells. We found that in senescent NHBE cells, telomerase activity, TERT mRNA levels, and telomere length were 60%, 2.7%, and 76% of those in proliferative cells, respectively. Using immunoprecipitation and TA cloning assays, we identified TpfeRNAa and TpfeRNAb, which were differentially expressed in proliferative and senescent NHBE cells. Blocking TpfeRNAb in senescent cells increased telomere length by 18% and boosted telomerase activity by tenfold, while ectopic expression of TpfeRNAb in proliferative decreased telomere length by 10%. These findings uncover TpfeRNAb as a key regulator of TERT activity to control telomere length, providing new insights into cellular senescence and aging-related diseases.

Primary idiopathic hyperhidrosis (PIH) is a poorly understood disorder characterized by excessive sweating. We identify a genetically defined subset of PIH associated with rare coding changes in voltage-gated Na ⁺ (Na _V ) channels. Whole-exome sequencing of hereditary PIH families revealed gene-level enrichment within the Na _V channel family, with SCN10A (Na _V 1.8) most strongly implicated. A knock-in mouse carrying the clinically observed Na _V 1.8 ^p.R14L substitution recapitulated excessive sweating. Na _V 1.8 was detected in a subset of postganglionic neurons in thoracic sympathetic ganglia in humans and mice, where p.R14L produced a gain-of-function profile that enhanced cholinergic responsiveness. Excessive sweating in mutant mice was reversible with Na _V channel inhibition, including clinically used agents and a Na _V 1.8-preferential compound. Together, these findings define a targetable neurocutaneous channelopathy underlying a subset of PIH and support a model in which excessive sweating arises from either gland-intrinsic dysfunction or altered sympathetic drive, motivating stratified therapeutic approaches.