Definition of a Multi-Omics Signature for Esophageal Adenocarcinoma Prognosis Prediction.

Esophageal cancer is a highly lethal malignancy, representing 5% of all cancer-related deaths. The two main subtypes are esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). While most research has focused on ESCC, few studies have analyzed EAC for transcriptional signatures linked to diagnosis or prognosis. In this study, we utilized single-cell RNA sequencing and bulk RNA sequencing to identify specific immune cell types that contribute to anti-tumor responses, as well as differentially expressed genes (DEGs). We have characterized transcriptional signatures, validated against a wide cohort of TCGA patients, that are capable of predicting clinical outcomes and the prognosis of EAC post-surgery with efficacy comparable to the currently accepted prognostic factors. In conclusion, our findings provide insights into the immune landscape and therapeutic targets of EAC, proposing novel immunological biomarkers for predicting prognosis, aiding in patient stratification for post-surgical outcomes, follow-up, and personalized adjuvant therapy decisions.

Inference of genomic lesions from single-cell RNA-seq in myeloma improves functional intraclonal and interclonal analysis.

ABSTRACT: Smoldering multiple myeloma (SMM) is an asymptomatic plasma cell (PC) neoplasm that may evolve with variable frequency into multiple myeloma (MM). SMM is initiated by chromosomal translocations involving the immunoglobulin heavy-chain locus or by hyperdiploidy and evolves through acquisition of additional genetic lesions. In this scenario, we aimed at establishing a reliable analysis pipeline to infer genomic lesions from transcriptomic analysis, by combining single-cell RNA sequencing (scRNA-seq) with B-cell receptor sequencing and copy number abnormality (CNA) analysis to identify clonal PCs at the genetic level along their specific transcriptional landscape. We profiled 20 465 bone marrow PCs derived from 5 patients with SMM/MM and unbiasedly identified clonal and polyclonal PCs. Hyperdiploidy, t(11;14), and t(6;14) were identified at the scRNA level by analysis of chimeric reads. Subclone functional analysis was improved by combining transcriptome with CNA analysis. As examples, we illustrate the different functional properties of a light-chain escape subclone in SMM and of different B-cell and PC subclones in a patient affected by Wäldenstrom macroglobulinemia and SMM. Overall, our data provide a proof of principle for inference of clinically relevant genotypic data from scRNA-seq, which in turn will refine functional annotation of the clonal architecture of PC dyscrasias.

MYTX-011: A pH-Dependent Anti-c-MET Antibody-Drug Conjugate Designed for Enhanced Payload Delivery to c-MET-Expressing Tumor Cells.

Advances in linker payload technology and target selection have been at the forefront of recent improvements in antibody-drug conjugate (ADC) design, leading to several approvals over the last decade. In contrast, the potential of novel ADC technologies to enhance payload delivery to tumors is relatively underexplored. We demonstrate that incorporation of pH-dependent binding in the antibody component of a c-mesenchymal-epithelial transition (MET)-targeting ADC (MYTX-011) can overcome the requirement for high c-MET expression on tumors, an innovation that has the potential to benefit a broader population of patients with lower c-MET levels. MYTX-011 drove fourfold higher net internalization than a non-pH-engineered parent ADC in non-small cell lung cancer (NSCLC) cells and showed increased cytotoxicity against a panel of cell lines from various solid tumors. A single dose of MYTX-011 showed at least threefold higher efficacy than a benchmark ADC in mouse xenograft models of NSCLC ranging from low to high c-MET expression. Moreover, MYTX-011 showed improved pharmacokinetics over parent and benchmark ADCs. In a repeat dose toxicology study, MYTX-011 exhibited a toxicity profile similar to other monomethyl auristatin E-based ADCs. These results highlight the potential of MYTX-011 for treating a broader range of patients with NSCLC with c-MET expression than other c-MET-targeting ADCs. A first-in-human study is ongoing to determine the safety, tolerability, and preliminary efficacy of MYTX-011 in patients with NSCLC (NCT05652868).

Multimaterial 3D Laser Printing of Cell-Adhesive and Cell-Repellent Hydrogels.

Here, a straightforward method is reported for manufacturing 3D microstructured cell-adhesive and cell-repellent multimaterials using two-photon laser printing. Compared to existing strategies, this approach offers bottom-up molecular control, high customizability, and rapid and precise 3D fabrication. The printable cell-adhesive polyethylene glycol (PEG) based material includes an Arg-Gly-Asp (RGD) containing peptide synthesized through solid-phase peptide synthesis, allowing for precise control of the peptide design. Remarkably, minimal amounts of RGD peptide (< 0.1 wt%) suffice for imparting cell-adhesiveness, while maintaining identical mechanical properties in the 3D printed microstructures to those of the cell-repellent, PEG-based material. Fluorescent labeling of the RGD peptide facilitates visualization of its presence in cell-adhesive areas. To demonstrate the broad applicability of the system, the fabrication of cell-adhesive 2.5D and 3D structures is shown, fostering the adhesion of fibroblast cells within these architectures. Thus, this approach allows for the printing of high-resolution, true 3D structures suitable for diverse applications, including cellular studies in complex environments.

MYTX-011: a pH-dependent anti-cMET antibody-drug conjugate designed for enhanced payload delivery to cMET expressing tumor cells.

Advances in linker payload technology and target selection have been at the forefront of recent improvements in antibody-drug conjugate (ADC) design, leading to several approvals over the last decade. In contrast, the potential of novel ADC technologies to enhance payload delivery to tumors is relatively underexplored. We demonstrate that incorporation of pH-dependent binding in the antibody component of a cMET targeting ADC (MYTX-011) can overcome the requirement for high cMET expression on tumors, an innovation that has the potential to benefit a broader population of patients with lower cMET levels. MYTX-011 drove four-fold higher net internalization than a non-pH engineered parent ADC in non-small cell lung cancer (NSCLC) cells and showed increased cytotoxicity against a panel of cell lines from various solid tumors. A single dose of MYTX-011 showed at least three-fold higher efficacy than a benchmark ADC in mouse xenograft models of NSCLC ranging from low to high cMET expression. Moreover, MYTX-011 showed improved pharmacokinetics over parent and benchmark ADCs. In a repeat dose toxicology study, MYTX-011 exhibited a toxicity profile similar to other MMAE-based ADCs. These results highlight the potential of MYTX-011 for treating a broader range of NSCLC patients with cMET expression than other cMET targeting ADCs. A first in human study is ongoing to determine the safety, tolerability, and preliminary efficacy of MYTX-011 in patients with NSCLC (NCT05652868).

Riboflavin-LSD1 axis participates in the in vivo tumor-associated macrophage morphology in human colorectal liver metastases.

Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment (TME). In colorectal liver metastasis (CLM), TAM morphology correlates with prognosis, with smaller TAMs (S-TAMs) conferring a more favorable prognosis than larger TAMs (L-TAMs). However, the metabolic profile of in vivo human TAM populations remains unknown. Multiparametric flow cytometry was used to freshly isolate S- and L-TAMs from surgically resected CLM patients (n = 14S-, 14L-TAMs). Mass spectrometry-based metabolomics analyses were implemented for the metabolic characterization of TAM populations. Gene expression analysis and protein activity were used to support the biochemical effects of the enzyme-substrate link between riboflavin and (lysine-specific demethylase 1A, LSD1) with TAM morphologies. L-TAMs were characterized by a positive correlation and a strong association between riboflavin and TAM morphologies. Riboflavin in both L-TAMs and in-vitro M2 polarized macrophages modulates LSD1 protein expression and activity. The inflammatory stimuli promoted by TNFα induced the increased expression of riboflavin transporter SLC52A3 and LSD1 in M2 macrophages. The modulation of the riboflavin-LSD1 axis represents a potential target for reprogramming TAM subtypes, paving the way for promising anti-tumor therapeutic strategies.

Breast implant surface topography triggers a chronic-like inflammatory response.

Breast implants are extensively employed for both reconstructive and esthetic purposes. However, the safety of breast implants with textured surfaces has been questioned, owing to a potential correlation with anaplastic large-cell lymphoma and the recurrence of breast cancer. This study investigates the immune response elicited by different prosthetic surfaces, focusing on the comparison between macrotextured and microtextured breast implants. Through the analysis of intraoperatively harvested periprosthetic fluids and cell culture experiments on surface replicas, we demonstrate that macrotextured surfaces elicit a more pronounced chronic-like activation of leucocytes and an increased release of inflammatory cytokines, in contrast to microtextured surfaces. In addition, in vitro fluorescent imaging of leucocytes revealed an accumulation of lymphocytes within the cavities of the macrotextured surfaces, indicating that the physical entrapment of these cells may contribute to their activation. These findings suggest that the topography of implant surfaces plays a significant role in promoting a chronic-like inflammatory environment, which could be a contributing factor in the development of lymphomas associated with a wide range of implantable devices.

Alteration of the immunophenotype and cytokine profiles in patients affected by neuroendocrine neoplasms.

PURPOSE: Neuroendocrine neoplasms (NENs) are tumors that arise from cells of the endocrine system and are most common in the gastrointestinal tract, the pancreas, and the lungs. Their incidence is rapidly increasing and the therapeutic options available are limited. METHODS: Since the immune system can interfere with tumor growth and response to therapy, using flow cytometry we investigated the immunophenotype in samples of peripheral blood leukocytes from patients with pancreatic (Pan-NENs) and pulmonary NENs (Lung-NENs). Moreover, we performed a multiplex analysis of 13 key cytokines and growth factors essential for the immune response in the plasma of NEN patients and controls. RESULTS: Patients presented with a higher percentage of granulocytes, a lower percentage of lymphocytes, and an increase in the granulocytes to lymphocytes ratio compared to healthy donors. These alterations were more marked in patients with metastasis. Somatostatin analogs (SSAs) restored the immunophenotype of patients to that seen in healthy donors. Finally, Pan-NEN patients showed a higher plasma concentration of IP-10, MCP-1, and IL-8 compared to healthy donors, suggesting a potential role for these cytokines as diagnostic biomarkers. CONCLUSION: This study highlighted differences in the immunophenotype of patients with Pan- and Lung-NENs compared to healthy individuals; these alterations were partially restored by therapy.

Single-Cell RNA Sequencing Reveals Metabolic Stress-Dependent Activation of Cardiac Macrophages in a Model of Dyslipidemia-Induced Diastolic Dysfunction.

BACKGROUND: Metabolic distress is often associated with heart failure with preserved ejection fraction (HFpEF) and represents a therapeutic challenge. Metabolism-induced systemic inflammation links comorbidities with HFpEF. How metabolic changes affect myocardial inflammation in the context of HFpEF is not known. METHODS: We found that ApoE knockout mice fed a Western diet recapitulate many features of HFpEF. Single-cell RNA sequencing was used for expression analysis of CD45+ cardiac cells to evaluate the involvement of inflammation in diastolic dysfunction. We focused bioinformatics analysis on macrophages, obtaining high-resolution identification of subsets of these cells in the heart, enabling us to study the outcomes of metabolic distress on the cardiac macrophage infiltrate and to identify a macrophage-to-cardiomyocyte regulatory axis. To test whether a clinically relevant sodium glucose cotransporter-2 inhibitor could ameliorate the cardiac immune infiltrate profile in our model, mice were randomized to receive the sodium glucose cotransporter-2 inhibitor dapagliflozin or vehicle for 8 weeks. RESULTS: ApoE knockout mice fed a Western diet presented with reduced diastolic function, reduced exercise tolerance, and increased pulmonary congestion associated with cardiac lipid overload and reduced polyunsaturated fatty acids. The main immune cell types infiltrating the heart included 4 subpopulations of resident and monocyte-derived macrophages, determining a proinflammatory profile exclusively in ApoE knockout- Western diet mice. Lipid overload had a direct effect on inflammatory gene activation in macrophages, mediated through endoplasmic reticulum stress pathways. Investigation of the macrophage-to-cardiomyocyte regulatory axis revealed the potential effects on cardiomyocytes of multiple inflammatory cytokines secreted by macrophages, affecting pathways such as hypertrophy, fibrosis, and autophagy. Finally, we describe an anti-inflammatory effect of sodium glucose cotransporter-2 inhibitor in this model. CONCLUSIONS: Using single-cell RNA sequencing , in a model of diastolic dysfunction driven by hyperlipidemia, we have determined the effects of metabolic distress on cardiac inflammatory cells, in particular on macrophages, and suggest sodium glucose cotransporter-2 inhibitors as potential therapeutic agents for the targeting of a specific phenotype of HFpEF.

Surface antibody changes protein corona both in human and mouse serum but not final opsonization and elimination of targeted polymeric nanoparticles.

BACKGROUND: Nanoparticles represent one of the most important innovations in the medical field. Among nanocarriers, polymeric nanoparticles (PNPs) attracted much attention due to their biodegradability, biocompatibility, and capacity to increase efficacy and safety of encapsulated drugs. Another important improvement in the use of nanoparticles as delivery systems is the conjugation of a targeting agent that enables the nanoparticles to accumulate in a specific tissue. Despite these advantages, the clinical translation of therapeutic approaches based on nanoparticles is prevented by their interactions with blood proteins. In fact, the so-formed protein corona (PC) drastically alters the biological identity of the particles. Adsorbed activated proteins of the complement cascade play a pivotal role in the clearance of nanoparticles, making them more easily recognized by macrophages, leading to their rapid elimination from the bloodstream and limiting their efficacy. Since the mouse is the most used preclinical model for human disease, this work compared human and mouse PC formed on untargeted PNPs (uPNPs) and targeted PNPs (tPNPs), paying particular attention to complement activation. RESULTS: Mouse and human serum proteins adsorbed differently to PNPs. The differences in the binding of mouse complement proteins are minimal, whereas human complement components strongly distinguish the two particles. This is probably due to the human origin of the Fc portion of the antibody used as targeting agent on tPNPs. tPNPs and uPNPs mainly activate complement via the classical and alternative pathways, respectively, but this pattern did not affect their binding and internalization in macrophages and only a limited consumption of the activity of the human complement system was documented. CONCLUSIONS: The results clearly indicate the presence of complement proteins on PNPs surface but partially derived from an unspecific deposition rather than an effective complement activation. The presence of a targeting antibody favors the activation of the classical pathway, but its absence allows an increased activation of the alternative pathway. This results in similar opsonization of both PNPs and similar phagocytosis by macrophages, without an impairment of the activity of circulating complement system and, consequently, not enhancing the susceptibility to infection.

The "Woggle" Technique for Venous Access Site Management: An Old Technique for a New Need.

BACKGROUND: Several closure devices are routinely used for percutaneous arterial access, while a relatively low number is available for the management of large bore venous accesses. The Woggle technique is a modification of the purse-string suture which was introduced several years ago in patients undergoing hemodialysis. METHODS: A population of 45 patients who underwent transvenous femoral structural heart interventions was retrospectively evaluated. The Woggle technique consists of a purge string suture with a collar to maintain the tension as stable over time and a suture lock to tighten the suture. RESULTS: Sheaths magnitude ranged from 8 French (F) to 14 F. A rapid post-procedural hemostasis was achieved in the whole population, and in 95% of cases, definite hemostasis was obtained after the first single release; the mean time of release was 302 ± 83 min. Although no relevant bleedings were reported, a significant reduction in hemoglobin levels was found in the whole population. This decrement was statistically significant only in the group with sheaths higher than 12 F. A single mild local hematoma was recorded in the group in which smaller sheaths were used. Seventy-two percent of patients were pre-treated with a dual antiplatelet therapy. CONCLUSIONS: The Woggle technique has shown to be a simple, effective, and safe approach for the management of large bore venous in percutaneous structural heart interventions.

Right Heart Catheterization: An Antecubital Vein Approach to Reduce Fluoroscopy Time, Radiation Dose, and Guidewires Need.

Antecubital access for right heart catheterization (RHC) is a widespread technique, even though there is a need to clarify if there are differences and significant advantages compared to proximal vein access. To pursue this issue, we retrospectively identified patients who underwent RHC in our clinic over a 7 year period (between January 2015 and December 2022). We revised demographic, anthropometric, and procedural data, including the fluoroscopy time, the radiation exposure, and the use of guidewires. The presence of any complications was also assessed. In patients with antecubital access, the fluoroscopy time and the radiation exposure were lower compared to proximal vein access (6 vs. 3 min, mean difference of 2 min, CI 95% 1-4 min, p < 0.001 and 61 vs. 30 cGy/m2, mean difference 64 cGy/m2, CI 95% 50-77, p < 0.001). The number of patients requiring the use of at least one guidewire was lower in the group undergoing RHC through antecubital access compared to proximal vein access (55% vs. 43%, p = 0.01). The feasibility was optimal, as just 0.9% of procedures switched from antecubital to femoral access, with a negligible rate of complications. The choice of the antecubital site exhibits advantages, e.g., a shorter fluoroscopy time, a reduced radiation dose, and a lower average number of guidewires used compared to proximal vein access.

Pattern of recurrence after stereotactic body radiotherapy for para-aortic oligo-recurrent prostate cancer, a multicentric analysis.

BACKGROUND: M1a disease represents an intermediate status between loco-regional relapse and bone metastatic disease. Metastasis directed therapy (MDT), through stereotactic body RT (SBRT) may be offered to patients, aiming to exclusively treat sites of macroscopic relapse and avoiding wide prophylactic treatment volumes. This appears as a viable treatment, especially after the rise of PSMA tailored treatment approaches. MATERIALS AND METHODS: Data about patients treated in two different institutions were retrieved from a prospectively collected dataset. All included patients were affected by oligo-recurrent M1a disease after definitive RT or radical prostatectomy, defined as ≤ 3 nodal lesions situated above aortic bifurcation and below renal arteries. Both castration resistant PCa (CRPC) and castration sensitive (CSPC) PCa patients were included. All imaging methods were allowed to detect recurrence (CT scan, Choline or PSMA PET/CT).All sites of recurrences were treated with SBRT. RESULTS: Median PFS was 10 months (95% CI 8-17). Twelve patients died, with a median OS of 114 months (95% CI 85-114). Out of the 83 recurrences, 2 (2.4%), 11 (13.25%), 36 (43.37%) and 15 (18%) patients had respectively prostate bed only, pelvic nodal, para-aortic or distant relapse. Furthermore, 19 (22.9%) patients experienced a biochemical only relapse with negative imaging at re-staging. DISCUSSION: MDT conferred a remarkable PFS outcome in a mixed cohort of CSPC and CRPC patients with m1a disease, with an optimal safety profile. Prospective trials are needed in order to compare MDT and ENRT for these patients, allowing to select the best treatment option.

Induced myocardial ischemia in candidates to liver transplantation without evidence of heart disease.

BACKGROUND: Coronary artery disease (CAD) is associated with perioperative liver transplantation (LT) mortality. In absence of a defined risk algorithm, we aimed to test whether stress echocardiography and coronary computed tomography angiography (CCTA) could detect CAD in end-stage liver disease (ESLD) patients without previous evidence of heart disease. METHODS: LT candidates ≥30 years underwent a cardiovascular (CV) assessment through stress echocardiography. CCTA was performed in patients ≥50 years with two or more CV risk factors (e.g. diabetes, CAD family history, dyslipidaemia). Coronary angiography (CAG) was scheduled when stress echocardiography and/or CCTA were positive. Sensibility, specificity, positive and negative predictive values of stress echocardiography and CCTA were assessed by numbers of coronary revascularization (true positives) and lack of acute coronary events over a mean follow-up of 3 years (true negatives). RESULTS: Stress echocardiography was performed in 273 patients, CCTA in 34 and CAG in 41. Eight patients had critical coronary lesions, and 19 not-critical lesions. Sensitivity, specificity, positive and negative predictive values were 50.0%, 90.2%, 13.3% and 98.4% for stress echocardiography and 100%, 76.7%, 36.4% and 100% for CCTA. Among 163 patients who underwent LT (57.6%), 16 died and 5 had major adverse CV events over a mean follow-up of 3 years. CONCLUSIONS: A very low prevalence of CAD in a selected population of ESLD at intermediate to high CV risk was found. A screening based on stress echocardiography and CCTA resulted in low incidence of post-LT acute coronary events in ELSD patients. CAD has no impact on mid-term survival.

Evaluation of Semen Self-Sampling Yield Predictors and CTC Isolation by Multi-Color Flow Cytometry for Liquid Biopsy of Localized Prostate Cancer.

Liquid biopsy (LB) for prostate cancer (PCa) detection could represent an alternative to biopsy. Seminal fluid (SF) is a source of PCa-specific biomarkers, as 40% of ejaculate derives from the prostate. We tested the feasibility of an SF-based LB by evaluating the yield of semen self-sampling in a cohort of >750 patients with clinically localized PCa. The overall SF collection yield was 18.2% (39% when considering only compliant patients), with about a half of the patients (53.15%) not consenting to SF donation. Independent favorable predictors for SF collection were younger age and lower prostate volume. We implemented a protocol to enrich prostate-derived cells by multi-color flow cytometry and applied it on SF and urine samples from 100 patients. The number of prostate-enriched cells (SYTO-16+ PSMA+ CD45-) was variable, with higher numbers of cells isolated from SF than urine (p value < 0.001). Putative cancer cells (EpCAMhigh) were 2% of isolated cells in both specimens. The fraction of EpCAMhigh cells over prostate-enriched cells (PSMA+) significantly correlated with patient age in both semen and urine, but not with other clinical parameters, such as Gleason Score, ISUP, or TNM stage. Hence, enumeration of prostate-derived cells is not sufficient to guide PCa diagnosis; additional molecular analyses to detect patient-specific cancer lesions will be needed.

High-Resolution Analysis of Mononuclear Phagocytes Reveals GPNMB as a Prognostic Marker in Human Colorectal Liver Metastasis.

Patients with colorectal liver metastasis (CLM) present with heterogenous clinical outcomes and improved classification is needed to ameliorate the therapeutic output. Macrophages (Mϕ) hold promise as prognostic classifiers and therapeutic targets. Here, stemming from a single-cell analysis of mononuclear phagocytes infiltrating human CLM, we identified two Mϕ markers associated with distinct populations with opposite clinical relevance. The invasive margin of CLM was enriched in pro-inflammatory monocyte-derived Mϕ (MoMϕ) expressing the monocytic marker SERPINB2, and a more differentiated population, tumor-associated Mϕ (TAM), expressing glycoprotein nonmetastatic melanoma protein B (GPNMB). SERPINB2+ MoMϕ had an early inflammatory profile, whereas GPNMB+ TAMs were enriched in pathways of matrix degradation, angiogenesis, and lipid metabolism and were found closer to the tumor margin, as confirmed by spatial transcriptomics on CLM specimens. In a cohort of patients, a high infiltration of SERPINB2+ cells independently associated with longer disease-free survival (DFS; P = 0.033), whereas a high density of GPNMB+ cells correlated with shorter DFS (P = 0.012) and overall survival (P = 0.002). Cell-cell interaction analysis defined opposing roles for MoMϕ and TAMs, suggesting that SERPINB2+ and GPNMB+ cells are discrete populations of Mϕ and may be exploited for further translation to an immune-based stratification tool. This study provides evidence of how multi-omics approaches can identify nonredundant, clinically relevant markers for further translation to immune-based patient stratification tools and therapeutic targets. GPNMB has been shown to set Mϕ in an immunosuppressive mode. Our high dimensional analyses provide further evidence that GPNMB is a negative prognostic indicator and a potential player in the protumor function of Mϕ populations.

Study of Seed Ageing in lpa1-1 Maize Mutant and Two Possible Approaches to Restore Seed Germination.

Phytic acid (PA) is a strong anti-nutritional factor with a key antioxidant role in countering reactive oxygen species. Despite the potential benefits of low phytic acid (lpa) mutants, the reduction of PA causes pleiotropic effects, e.g., reduced seed germination and viability loss related to seed ageing. The current study evaluated a historical series of naturally aged seeds and showed that lpa1-1 seeds aged faster as compared to wildtype. To mimic natural ageing, the present study set up accelerated ageing treatments at different temperatures. It was found that incubating the seeds at 57 °C for 24 h, the wildtype germinated at 82.4% and lpa1-1 at 40%. The current study also hypothesized two possible solutions to overcome these problems: (1) Classical breeding was used to constitute synthetic populations carrying the lpa1-1 mutation, with genes pushing anthocyanin accumulation in the embryo (R-navajo allele). The outcome showed that the presence of R-navajo in the lpa1-1 genotype was not able to improve the germinability (-20%), but this approach could be useful to improve the germinability in non-mutant genotypes (+17%). (2) In addition, hydropriming was tested on lpa1-1 and wildtype seeds, and germination was improved by 20% in lpa1-1, suggesting a positive role of seed priming in restoring germination. Moreover, the data highlighted metabolic differences in the metabolome before and after hydropriming treatment, suggesting that the differences in germination could also be mediated by differences in the metabolic composition induced by the mutation.

Single-Cell RNA Sequencing for the Detection of Clonotypic V(D)J Rearrangements in Multiple Myeloma.

Multiple myeloma (MM) has a highly heterogeneous genetic background, which complicates its molecular tracking over time. Nevertheless, each MM patient's malignant plasma cells (PCs) share unique V(D)J rearranged sequences at immunoglobulin loci, which represent ideal disease biomarkers. Because the tumor-specific V(D)J sequence is highly expressed in bulk RNA in MM patients, we wondered whether it can be identified by single-cell RNA sequencing (scRNA-seq). To this end we analyzed CD138+ cells purified from bone marrow aspirates of 19 samples with PC dyscrasias by both a standard method based on bulk DNA and by an implementation of the standard 10x Genomics protocol to detect expressed V(D)J sequences. A dominant clonotype was easily identified in each sample, accounting on average for 83.65% of V(D)J-rearranged cells. Compared with standard methods, scRNA-seq analysis proved highly concordant and even more effective in identifying clonal productive rearrangements, by-passing limitations related to the misannealing of consensus primers in hypermutated regions. We next validated its accuracy to track 5 clonal cells with absolute sensitivity in a virtual sample containing 3180 polyclonal cells. This shows that single-cell V(D)J analysis may be used to find rare clonal cells, laying the foundations for functional single-cell dissection of minimal residual disease.

The Potential of Low Phytic Acid1-1 Mutant in Maize (Zea mays L.): A Sustainable Solution to Non-Renewable Phosphorus.

BACKGROUND: Phosphorus is an essential component of fertilizers and feed and in recent decades has become one of the main sustainability issues as a non-renewable resource. In plant seeds, the main reserve of phosphorus is phytic acid, a strong anti-nutritional factor for monogastrics and a pollutant of cultivated lands. The reduction of phytic acid in cereal seeds has become a major challenge in breeding programs to increase the nutritional quality of foods and feeds and to improve the environmental phosphorus sustainability in agriculture. In maize (Zea mays L.), four low phytic acid (lpa) mutations have been isolated and lpa1-1 is the most promising. However, the reduction of phytic acid in lpa1-1 leads to many adverse pleiotropic effects on the seed and in general on plant performance. A seed weight reduction and a consequent yield loss were previously described in this mutant. METHOD: In this work, a field experiment to study seed weight and yield was conducted for two years in two different genetic backgrounds (B73 and B73/Mo17). Furthermore, the greater susceptibility of lpa1-1 to drought stress was also investigated: a dedicated field experiment was set up and measurements were carried out under optimal water conditions and moderate drought stress. RESULTS: From the first experiment it emerges that under high-input conditions, lpa1-1 seems to have comparable or even better yield than the relative control. The main problem of this mutant remains the reduced field emergence (~40%). In the study of drought stress it was found that the increased sensitivity in the mutant is mainly caused by an altered stomatal regulation, but not by a less developed root system, as previously reported. When the stress occurred, the parameters measured did not significantly change in the wild-type, while they dropped in the mutant: the net photosynthesis decreased by 58%, the transpiration rate by 63% and the stomatal conductance by 67%. CONCLUSIONS: Some possible solutions have been proposed, with the aim of developing a commercial variety, which remains the main goal to exploit the nutritional benefits of low phytic acid mutants.