ABSTRACT
Vault RNAs (vRNAs) are evolutionarily conserved small non-coding RNAs transcribed by RNA polymerase lll. Initially described as components of the vault particle, they have since also been described as noncanonical miRNA precursors and as riboregulators of autophagy. As central molecules in these processes, vRNAs have been attributed numerous biological roles including regulation of cell proliferation and survival, response to viral infections, drug resistance, and animal development. Yet, their impact to mammalian physiology remains largely unexplored. To study vault RNAs in vivo, we generated a mouse line with a conditional Vaultrc5 loss of function allele. Because Vaultrc5 is the sole murine vRNA, this allele enables the characterization of the physiological requirements of this conserved class of small regulatory RNAs in mammals. Using this strain, we show that mice constitutively null for Vaultrc5 are viable and histologically normal but have a slight reduction in platelet counts pointing to a potential role for vRNAs in hematopoiesis. This work paves the way for further in vivo characterizations of this abundant but mysterious RNA molecule. Specifically, it enables the study of the biological consequences of constitutive or lineage-specific Vaultrc5 deletion and of the physiological requirements for an intact Vaultrc5 during normal hematopoiesis or in response to cellular stresses such as oncogene expression, viral infection, or drug treatment.
ABSTRACT
An Artificial Metalloenzyme (ArM) built employing the streptavidin-biotin technology has been used for the enantioselective synthesis of binaphthyls by means of asymmetric Suzuki-Miyaura cross-coupling reactions. Despite its success, it remains a challenge to understand how the length of the biotin cofactors or the introduction of mutations to streptavidin leads the preferential synthesis of one atropisomer over the other. In this study, we apply an integrated computational modeling approach, including DFT calculations, protein-ligand dockings and molecular dynamics to rationalize the impact of mutations and length of the biotion cofactor on the enantioselectivities of the biaryl product. The results unravel that the enantiomeric differences found experimentally can be rationalized by the disposition of the first intermediate, coming from the oxidative addition step, and the entrance of the second substrate. The work also showcases the difficulties facing to control the enantioselection when engineering ArM to catalyze enantioselective Suzuki-Miyaura couplings and how the combination of DFT calculations, molecular dockings and MD simulations can be used to rationalize artificial metalloenzymes.
Subject(s)
Density Functional Theory , Molecular Dynamics Simulation , Streptavidin , Stereoisomerism , Streptavidin/chemistry , Streptavidin/metabolism , Catalysis , Biotin/chemistry , Biotin/analogs & derivatives , Ligands , Molecular Docking Simulation , Metalloproteins/chemistry , Metalloproteins/metabolismABSTRACT
INTRODUCTION: The synthesis of the periprosthetic capsule during implant-based breast reconstruction is the result of a coordinate cascade of inflammatory events ending in a fibrous tissue deposition around the expander or implant. Although the development of small volumes of fluid is one of the complications of prosthetic-based breast reconstruction, the characterization of the periprosthetic effusions coupled with the micro-textured devices, that have been recently introduced after the recall of macro-textured ones, is still lacking. The investigation of these periprosthetic effusions and paired capsules in terms of immunological content were the primary and secondary aims of the present study, respectively. METHODS: For this, 68 women, 41 of whom had periprosthetic effusions at the time of expander replacement with implant, were recruited. For each case, capsule and healthy dermal tissues were taken and for women with periprosthetic effusion, peripheral blood was also collected. Periprosthetic effusions and peripheral blood were characterized by cytometry while capsules and dermal tissues by immunohistochemistry and Nanostring analysis. RESULTS: The results showed an increase of Th1, Th2 lymphocytes and a HLA-DR+bright CD16+ cells (likely representing monocytes-derived macrophages) in periprosthetic effusions in respect to peripheral blood. These pro-inflammatory cells were counterbalanced by the gain of suppressive CD4 Treg cells. In the corresponding capsules, immunohistochemistry revealed the absence of Th1 cells and the presence of tissutal FOXP3 Treg. No significant difference in expression of inflammatory-related genes between capsules and dermal tissues was present. CONCLUSIONS: These results suggest the presence of a Treg-controlled inflammation in both periprosthetic effusions and capsules.
Subject(s)
Breast Implants , Breast Neoplasms , Mammaplasty , Female , Humans , Mammaplasty/methods , InflammationABSTRACT
BACKGROUND: As in most solid cancers, the emergence of cells with oncogenic mutations in the mammary epithelium alters the tissue homeostasis. Some soluble factors, such as TGFß, potently modify the behavior of healthy stromal cells. A subpopulation of cancer-associated fibroblasts expressing a TGFß target, the SNAIL1 transcription factor, display myofibroblastic abilities that rearrange the stromal architecture. Breast tumors with the presence of SNAIL1 in the stromal compartment, and with aligned extracellular fiber, are associated with poor survival prognoses. METHODS: We used deep RNA sequencing and biochemical techniques to study alternative splicing and human tumor databases to test for associations (correlation t-test) between SNAIL1 and fibronectin isoforms. Three-dimensional extracellular matrices generated from fibroblasts were used to study the mechanical properties and actions of the extracellular matrices on tumor cell and fibroblast behaviors. A metastatic mouse model of breast cancer was used to test the action of fibronectin isoforms on lung metastasis. RESULTS: In silico studies showed that SNAIL1 correlates with the expression of the extra domain A (EDA)-containing (EDA+) fibronectin in advanced human breast cancer and other types of epithelial cancers. In TGFß-activated fibroblasts, alternative splicing of fibronectin as well as of 500 other genes was modified by eliminating SNAIL1. Biochemical analyses demonstrated that SNAIL1 favors the inclusion of the EDA exon by modulating the activity of the SRSF1 splicing factor. Similar to Snai1 knockout fibroblasts, EDA- fibronectin fibroblasts produce an extracellular matrix that does not sustain TGFß-induced fiber organization, rigidity, fibroblast activation, or tumor cell invasion. The presence of EDA+ fibronectin changes the action of metalloproteinases on fibronectin fibers. Critically, in an mouse orthotopic breast cancer model, the absence of the fibronectin EDA domain completely prevents lung metastasis. CONCLUSIONS: Our results support the requirement of EDA+ fibronectin in the generation of a metastasis permissive stromal architecture in breast cancers and its molecular control by SNAIL1. From a pharmacological point of view, specifically blocking EDA+ fibronectin deposition could be included in studies to reduce the formation of a pro-metastatic environment.
Subject(s)
Breast Neoplasms , Lung Neoplasms , Animals , Female , Humans , Mice , Alternative Splicing , Breast Neoplasms/genetics , Fibronectins/genetics , Fibronectins/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Serine-Arginine Splicing Factors/genetics , Serine-Arginine Splicing Factors/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
Argonaute 2 (AGO2) is a cytoplasmic component of the miRNA pathway, with essential roles in development and disease. Yet little is known about its regulation in vivo. Here we show that in quiescent mouse splenocytes, AGO2 localizes almost exclusively to the nucleus. AGO2 subcellular localization is modulated by the Pi3K-AKT-mTOR pathway, a well-established regulator of quiescence. Signaling through this pathway in proliferating cells promotes AGO2 cytoplasmic accumulation, at least in part by stimulating the expression of TNRC6, an essential AGO2 binding partner in the miRNA pathway. In quiescent cells in which mTOR signaling is low, AGO2 accumulates in the nucleus, where it binds to young mobile transposons co-transcriptionally to repress their expression via its catalytic domain. Our data point to an essential but previously unrecognized nuclear role for AGO2 during quiescence as part of a genome-defense system against young mobile elements and provide evidence of RNA interference in the soma of mammals.
Subject(s)
Argonaute Proteins , MicroRNAs , Phosphatidylinositol 3-Kinases , Animals , Mice , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Cell Line , Mammals/genetics , MicroRNAs/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , RNA Interference , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolismABSTRACT
Importance: Melanoma guidelines recommend surgical excision with 10-mm margins for T1 melanoma. However, this procedure may be problematic at sites close to critical structures such as the scalp, face, external genitalia, acral, periumbilical, and perineal areas. Objective: To compare outcomes of wide (10-mm margins) vs narrow (5-mm margins) excision in patients with T1a melanoma near critical structures. Design, Setting, and Participants: This cohort study was a retrospective comparison of 1341 consecutive patients aged 18 years or older from the National Cancer Institute of Milan, Italy, diagnosed between 2001 and 2020 with T1a cutaneous melanoma close to critical structures who accepted wide excision vs narrow excision. Exposures: Local recurrence and melanoma-specific mortality (MSM) rates with 5-mm vs 10-mm excision margins. Main Outcomes and Measures: The primary aim of the study was to ascertain whether a narrower (5-mm) vs wider (10-mm) excision margin was associated with local recurrence and MSM. The secondary aim was to compare the need for reconstructive surgery in the groups defined by excision margin width. Between April 28 and August 7, 2022, associations were assessed by weighted Cox and Fine-Gray univariable and multivariable models. Results: A total of 1179 patients met the inclusion criteria (median [IQR] age, 50.0 [39.5-63.0] years; female, 610 [51.7%]; male, 569 [49.3%]). Six hundred twenty-six patients (53.1%) received a wide excision (434 [69.3%] with linear repair and 192 [30.7%] with flap or graft reconstruction) and 553 (46.9%) received a narrow excision (491 [88.8%] with linear repair and 62 [11.2%] with flap or graft reconstruction). The weighted 10-year MSM was 1.8% (95% CI, 0.8%-4.2%) in the wide group and 4.2% (95% CI, 2.2%-7.9%) in the narrow group; the weighted 10-year local recurrence rate was 5.7% (95% CI, 3.9%-8.3%) in the wide group and 6.7% (95% CI, 4.7%-9.5%) in the narrow group. Breslow thickness greater than 0.4 mm (subdistribution hazard ratio [sHR] for 0.6 vs 0.4 mm, 2.42; 95% CI, 1.59-3.68; P < .001) and mitotic rate greater than 1/mm2 (sHR for a single increment, 3.35; 95% CI, 2.59-4.32; P < .001) were associated with worse MSM. Multivariable analysis showed that acral lentiginous melanoma, lentigo maligna melanoma, and increasing Breslow thickness were associated with a higher incidence of local recurrence. Conclusions and Relevance: The study's findings suggest that local excision with 5-mm margins for T1a melanoma may not be associated with an increased risk of local recurrence. Breslow thickness greater than 0.4 mm, mitotic rate greater than 1/mm2, and acral lentiginous melanoma and lentigo maligna melanoma subtypes were associated with a higher risk of recurrence. These findings may be useful for future melanoma treatment guidelines.
Subject(s)
Hutchinson's Melanotic Freckle , Melanoma , Sexual and Gender Minorities , Skin Neoplasms , Humans , Male , Female , Middle Aged , Melanoma/surgery , Skin Neoplasms/surgery , Margins of Excision , Cohort Studies , Retrospective Studies , Homosexuality, Male , Neoplasm Recurrence, Local/epidemiology , Melanoma, Cutaneous MalignantABSTRACT
INTRODUCTION: During the last few years it has been shown that an anaplastic T cell lymphoma can develop as a rare and late sequelae of implant-based breast reconstruction. This malignancy was recognized in the 2017 by WHO and named breast implant associated anaplastic large T cell lymphoma (BIA-ALCL). BIA-ALCL usually presents as abundant effusion around the implant, thus, in addition to cytology smears, its diagnosis also requires immunohistochemistry, T cells clonality and cytometry. Due to the increasing attention of clinicians, it is likely that the number of the BIA-ALCL suspected cases will grow in the future, implying the necessity of a reliable and cost-effective diagnostic procedure. METHODS: To achieve this goal, we retrospectively analyzed the results of laboratory investigations performed at our Institute (Fondazione IRCCS Istituto Nazionale dei Tumori Milan, Italy) on 44 effusions obtained from 31 women suspected for BIA-ALCL. RESULTS: Through cytology, eight out of 44 effusions showed the presence of BIA-ALCL cells. Lymphoma cells were than confirmed in seven samples by immunohistochemistry and/or T cell clonality and/or cytometry. Overall, cytology showed 100% sensitivity, 97% specificity and positive and negative predictive values of 87.5% and 100% respectively. Further analyses were particularly useful in effusions showing small percentages of BIA-ALCL cells. Moreover, an extended cytometric profile that can be applied when fast confirmation of the cytologic result is required was also identified. CONCLUSIONS: Our results evidenced a central role of cytopathology in the management of BIA-ALCL suspected effusions and suggested that further laboratory investigations might be applied only in cases showing atypical/activated lymphoid cells through cytology.
Subject(s)
Breast Implantation , Breast Implants , Breast Neoplasms , Lymphoma, Large-Cell, Anaplastic , Female , Humans , Breast Implants/adverse effects , Lymphoma, Large-Cell, Anaplastic/diagnosis , Lymphoma, Large-Cell, Anaplastic/etiology , Lymphoma, Large-Cell, Anaplastic/pathology , Retrospective Studies , Breast Implantation/methods , Breast Neoplasms/pathologyABSTRACT
BACKGROUND: Breast reconstruction has become a standard of care in patients undergoing mastectomy, greatly improving their quality of life. An increasing number of patients-reported outcome measurements (PROMs) have been developed over the years to better analyze patients' subjective overall experience. BREAST-Q is the PROMs for breast surgery introduced in our practice to assess patients' experiences when undergoing implant-based breast reconstruction and radiotherapy along with the use of Peak Plasma Blade and acellular dermal matrix. METHODS: The pre-operative version of the Reconstruction BREAST-Q was administered to all 88 patients enrolled between December 2017 and December 2018 in the Plastic Reconstructive Surgery Unit at Fondazione IRCCS Istituto Nazionale Tumori Milano through person-to-person interviews, while the post-operative version was administered to the 75 patients who completed a 12-month follow-up (four patients died during one-year follow-up and nine patients had major complications). The survey areas highlighted were: satisfaction with breast, psychosocial well-being, physical well-being and sexual well-being. RESULTS: From BREAST-Q questions regarding Satisfaction With The Appearance Of The Breast and Psychosocial Well-Being outcomes showed significant improvement from pre-operative data, as well as with Satisfaction With Overall Care, with the exception of Physical Well-Being Chest. CONCLUSIONS: BREAST-Q allows the assessment of patients' perception, not only for surgical results, but also for the overall experience with surgeons and medical staff.The women enrolled in our study reported an overall good patient satisfaction in most of the analyzed fields.
Subject(s)
Breast Neoplasms , Mammaplasty , Female , Humans , Mastectomy/methods , Quality of Life , Breast Neoplasms/radiotherapy , Breast Neoplasms/surgery , Mammaplasty/methods , Patient Satisfaction , Patient Reported Outcome MeasuresABSTRACT
BACKGROUND: Implant-based breast reconstruction in the setting of radiotherapy often leads to higher complications rates (mainly capsular contracture and wound dehiscence) and poor cosmetic outcomes. We hypothesized that the combination of pulsed-electron avalanche knife (PEAK) PlasmaBlade (a pulsed radiofrequency electrosurgery) and acellular dermal matrix Veritas® in postmastectomy radiotherapy implant-based breast reconstruction could result in lower complications rate, better reconstructive results, and patient satisfaction. METHODS: A prospective observational study focused on the use of PEAK PlasmaBlade in implant-based breast reconstruction and radiotherapy was carried out in the Plastic Reconstructive Surgery Unit at Fondazione IRCCS Istituto Nazionale Tumori Milano between December 2017 and 2019 (2017-2018: enrollment; 2018-2019: follow-up). Patient demographics were queried and complication rates and patient and surgeon satisfaction were assessed. RESULTS: A total of 88 patients were enrolled; 2 patients received bilateral reconstruction, leading to a total of 90 procedures. Sixty-two women received contralateral symmetrization. Seroma was the most frequent minor complication (8.8%); implant exposure was the most recorded among major complications (5.5%). Preoperative lipofilling was the most substantial protective factor for preventing complications (p < 0.001). A significant association between capsular thermal damage thickness and the type of electrosurgery used (traditional electrosurgery vs PEAK PlasmaBlade) was observed, with lower values with PEAK PlasmaBlade (p < 0.0001). CONCLUSIONS: Our protocol results in low rates of surgical complications and a high level of patient and surgeon satisfaction although longer follow-up is needed.
Subject(s)
Breast Neoplasms , Mammaplasty , Humans , Female , Breast Neoplasms/radiotherapy , Breast Neoplasms/surgery , Electrons , Mastectomy/adverse effects , Retrospective Studies , Mammaplasty/adverse effects , Mammaplasty/methods , Postoperative Complications/etiology , Follow-Up StudiesABSTRACT
The covalent insertion of a cobalt heme into the cavity of an artificial protein named alpha Rep (αRep) leads to an artificial cobalt hemoprotein that is active as a catalyst not only for the photo-induced production of H2, but also for the reduction of CO2 in a neutral aqueous solution. This new artificial metalloenzyme has been purified and characterized by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS), circular dichroism, and UltraViolet-Visible spectroscopy. Using theoretical experiments, the structure of this biohybrid and the positioning of the residues near the metal complex were examined, which made it possible to complete the coordination of the cobalt ion by an axial glutamine Gln283 ligand. While the Co(III)-porphyrin catalyst alone showed weak catalytic activity for both reactions, 10 times more H2 and four times more CO2 were produced when the Co(III)-porphyrin complex was buried in the hydrophobic cavity of the protein. This study thus provides a solid basis for further improvement of these biohybrids using well-designed modifications of the second and outer coordination sphere by site-directed mutagenesis of the host protein.
Subject(s)
Coordination Complexes , Hemeproteins , Porphyrins , Cobalt/chemistry , Carbon Dioxide/chemistry , Coordination Complexes/chemistry , Catalysis , Hydrogen/chemistryABSTRACT
Many biological systems obtain their activity by the inclusion of metalloporphyrins into one or several binding pockets. However, decoding the molecular mechanism under which these compounds bind to their receptors is something that has not been widely explored and is a field with open questions. In the present work, we apply computational techniques to unravel and compare the mechanisms of two heme-binding systems, concretely the HasA hemophores from Gram negative bacteria Serratiamarcescens (HasAsm) and Yersinia pestis (HasAyp). Despite the high sequence identity between both systems, the comparison between the X-ray structures of their apo and holo forms suggests different heme-binding mechanisms. HasAyp has extremely similar structures for heme-free and heme-bound forms, while HasAsm presents a very large displacement of a loop that ultimately leads to an additional coordination to the metal with respect to HasAyp. We combined Gaussian accelerated molecular dynamics simulations (GaMDs) in explicit solvent and protein-ligand docking optimized for metalloligands. GaMDs were first carried out on heme-free forms of both hemophores. Then, protein-ligand dockings of the heme were performed on cluster representatives of these simulations and the best poses were then subjected to a new series of GaMDs. A series of analyses reveal the following: (1) HasAyp has a conformational landscape extremely similar between heme-bound and unbound states with no to limited impact on the binding of the cofactor, (2) HasAsm presents as a slightly broader conformational landscape in its apo state but can only visit conformations similar to the X-ray of the holo form when the heme has been bound. Such behavior results from a complex cascade of changes in interactions that spread from the heme-binding pocket to the flexible loop previously mentioned. This study sheds light on the diversity of molecular mechanisms of heme-binding and discusses the weight between the pre-organization of the receptor as well as the induced motions resulting in association.
Subject(s)
Bacterial Proteins , Heme , Ligands , Bacterial Proteins/chemistry , Heme/chemistry , Carrier Proteins/chemistry , Molecular Dynamics Simulation , Protein ConformationABSTRACT
The direct oxidation of benzene into phenol using molecular oxygen at very mild temperatures can be promoted in the presence of the copper complex TpBr3Cu(NCMe) in the homogeneous phase in the presence of ascorbic acid as the source of protons and electrons. The stoichiometric nature, relative to copper, of this transformation prompted a thorough DFT study in order to understand the reaction pathway. As a result, the dinuclear species TpBr3CuII(µ-Oâ¢)(µ-OH)CuIITpBr3 is proposed as the relevant structure which is responsible for activating the arene C-H bond leading to phenol formation.
ABSTRACT
BACKGROUND: The early developmental interventions might be designed with a preventative approach to improving the development of at-risk preterm infants. The present study aimed to evaluate the effectiveness of an early physiotherapy intervention on preterm infants' motor and global development, and on parents' stress index. METHODS: 48 infants were enrolled and randomized into two groups. Infants allocated to the intervention group received an early physiotherapy intervention, based on parental education sessions and tactile and kinesthetic stimulation during the NICU period, as well as a home-based activity program. The intervention commenced after 32 weeks post-menstrual age and ended at 2 months corrected age. Infants allocated to the control group received the usual care based on the NIDCAP-care. RESULTS: No differences were found between groups on the Alberta Infant Motor Scale at 2- or 8-months corrected age. Infants in the intervention group showed more optimal fine motor, problem-solving, personal-social, and communication development at 1 month corrected age. CONCLUSIONS: The results showed no effect on the early physiotherapy intervention. Results might be related to the dose or intensity of the intervention, but also to the poor parental compliance. CLINICALTRIALS: gov NCT03313427.
ABSTRACT
Off-target effects are well established confounders of CRISPR negative selection screens that impair the identification of essential genomic loci. In particular, non-coding regulatory elements and repetitive regions are often difficult to target with specific gRNAs, effectively precluding the unbiased screening of a large portion of the genome. To address this, we developed CRISPR Specificity Correction (CSC), a computational method that corrects for the effect of off-targeting on gRNA depletion. We benchmark CSC with data from the Cancer Dependency Map and show that it significantly improves the overall sensitivity and specificity of viability screens while preserving known essentialities, particularly for genes targeted by highly promiscuous gRNAs. We believe this tool will further enable the functional annotation of the genome as it represents a robust alternative to the traditional filtering strategy of discarding unspecific guides from the analysis. CSC is an open-source software that can be seamlessly integrated into current CRISPR analysis pipelines.
Subject(s)
RNA, Guide, Kinetoplastida/metabolism , CRISPR-Cas Systems/genetics , CRISPR-Cas Systems/physiology , Computational Biology/methods , Gene Editing , Humans , RNA, Guide, Kinetoplastida/genetics , SoftwareABSTRACT
OBJECTIVE: To compare and evaluate the results and suitability of two different approaches to the treatment of post-conization International Federation of Gynaecology and Obstetrics (FIGO) stage IA1 cervical carcinoma: a more radical approach, directly scheduling a second surgery versus a more conservative one, which consists of performing a cotest (PAP plus HPV-test) in a follow-up visit and deciding whether to apply a second surgery on the basis of the results. STUDY DESIGN: Retrospective descriptive study including 144 cases of stage IA1 cervical carcinoma diagnosed after a loop electrosurgical excisional procedure (conization), between 1987 and 2019 in the Mother-and-Child University Hospital of Gran Canaria (Spain). Selected patients were split into two groups for analysis: patients directly undergoing a second surgical intervention (hysterectomy or re-conization) after diagnosis and patients who were followed-up before making a decision whether to schedule a second surgery or continue to follow-up. RESULTS: 75% of women directly receiving a second surgical intervention (no post-conization follow-up) underwent hysterectomy, while 25% underwent re-conization. Histological outcomes from hysterectomized patients showed 65% negative results for intraepithelial lesions, 9% low-grade squamous intraepithelial lesions (LSIL), 16% high-grade squamous intraepithelial lesions (HSIL) and only 10.5% confirmed invasive lesions: hysterectomy complication rate was 7%. Histological studies from women subjected to re-conization showed 32% negative results, 37% LSIL, 5% HSIL and 26% malignancy. In the group of patients who were followed-up after diagnosis, 8.8% needed a second intervention; none of them showed negative histological results, while 100% hysterectomized and 25% patients with re-conization showed HSIL. No unnecessary hysterectomy procedures were conducted in this group. HPV-16 was the most common genotype in both groups. CONCLUSION: Conization proved to be a suitable alternative to hysterectomy as a treatment for post-conization stage IA1 cervical cancer. Our results showed that 65% hysterectomy procedures conducted without previously monitoring for residual disease corresponded to negative results and were therefore, unnecessary. We conclude that confirmation of the presence of residual disease by using cotest is essential to make a decision on further treatment and that a conservative management is often possible and, in our opinion, preferable.
Subject(s)
Carcinoma, Squamous Cell , Uterine Cervical Neoplasms , Carcinoma, Squamous Cell/surgery , Conization , Epithelial Cells , Female , Humans , Hysterectomy , Pregnancy , Retrospective Studies , Uterine Cervical Neoplasms/surgeryABSTRACT
BACKGROUND: Prognostic parameters in sentinel node (SN)-positive melanoma are important indicators to identify patients at high risk of recurrence who should be candidates for adjuvant therapy. We aimed to evaluate the presence of melanoma cells beyond the SN capsule-extranodal extension (ENE)-as a prognostic factor in patients with positive SNs. METHODS: Data from 1,047 patients with melanoma and positive SNs treated from 2001 to 2020 at the Istituto Nazionale dei Tumori in Milano, Italy, were retrospectively investigated. Kaplan-Meier survival and crude cumulative incidence of recurrence curves were estimated. A multivariable logistic model was used to investigate the association between ENE and selected predictive factors. Cox models estimated the effect of the selected predictors on survival endpoints. RESULTS: Median follow-up was 69 months. The 5-year overall survival rate was 62.5% and 71.7% for patients with positive SNs with and without ENE, respectively. The 5-year disease-free survival rate was 54.0% and 64.0% for patients with positive SNs with and without ENE, respectively. The multivariable logistic model showed that age, size of the main metastatic focus in the SN, and numbers of metastatic non-SNs were associated with ENE (all P<.0001). The multivariable Cox regression models showed the estimated prognostic effects of ENE associated with age, ulceration, size of the main metastatic focus in the SN, and number of metastatic non-SNs (all P<.0001) on disease-free survival and overall survival. CONCLUSIONS: ENE was a significant prognostic factor in patients with positive-SN melanoma. This parameter may be useful in clinical practice as a selection criterion for adjuvant treatment in patients with stage IIIA disease with a tumor burden <1 mm in the SN. We recommend its inclusion as an independent prognostic determinant in future updates of melanoma guidelines.
Subject(s)
Melanoma , Skin Neoplasms , Extranodal Extension , Humans , Lymph Node Excision , Lymph Nodes/pathology , Lymphatic Metastasis/pathology , Melanoma/pathology , Prognosis , Retrospective Studies , Sentinel Lymph Node Biopsy , Skin Neoplasms/pathologyABSTRACT
Early-onset severe spinocerebellar ataxia 42 with neurodevelopmental deficits (SCA42ND, MIM#604065) is an ultrarare autosomal dominant syndrome related to de novo CACNA1G gain-of-function pathogenic variants. All patients with SCA42ND show cerebellar atrophy and/or hypoplasia on neuroimaging and share common features such as dysmorphic features, global developmental delay, and axial hypotonia, all manifesting within the first year of life. To date, only 10 patients with SCA42ND have been reported with functionally confirmed gain-of-function variants, bearing either of two recurrent pathogenic variants. We describe a girl with congenital ataxia, without epilepsy, and a de novo p.Ala961Thr pathogenic variant in CACNA1G. We review the published subjects with the aim of better characterizing the dysmorphic features that may be crucial for clinical recognition of SCA42ND. Cerebellar atrophy, together with digital anomalies, particularly broad thumbs and/or halluces, should lead to clinical suspicion of this disease. We describe the first pharmacological attempt to treat a patient with SCA42ND using zonisamide, an antiepileptic drug with T-type channel blocker activity, in an off-label indication using an itemized study protocol. No efficacy was observed at the dose tested. However, without pharmacological treatment, she showed a positive evolution in neurodevelopment during the follow-up.
Subject(s)
Calcium Channels, T-Type/genetics , Epilepsy/genetics , Muscle Hypotonia/genetics , Spinocerebellar Ataxias/genetics , Age of Onset , Alleles , Child, Preschool , Epilepsy/complications , Epilepsy/diagnostic imaging , Epilepsy/drug therapy , Female , Gain of Function Mutation/genetics , Humans , Infant , Male , Muscle Hypotonia/complications , Muscle Hypotonia/diagnostic imaging , Muscle Hypotonia/drug therapy , Mutation , Pedigree , Phenotype , Spinocerebellar Ataxias/complications , Spinocerebellar Ataxias/diagnostic imaging , Spinocerebellar Ataxias/drug therapy , Zonisamide/administration & dosageABSTRACT
With a large amount of research dedicated to decoding how metallic species bind to proteins, in silico methods are interesting allies for experimental procedures. To date, computational predictors mostly work by identifying the best possible sequence or structural match of the target protein with metal-binding templates. These approaches are fundamentally focused on the first coordination sphere of the metal. Here, we present the BioMetAll predictor that is based on a different postulate: the formation of a potential metal-binding site is related to the geometric organization of the protein backbone. We first report the set of convenient geometric descriptors of the backbone needed for the algorithm and their parameterization from a statistical analysis. Then, the successful benchmark of BioMetAll on a set of more than 90 metal-binding X-ray structures is presented. Because BioMetAll allows structural predictions regardless of the exact geometry of the side chains, it appears extremely valuable for systems whose structures (either experimental or theoretical) are not optimal for metal-binding sites. We report here its application on three different challenging cases: (i) the modulation of metal-binding sites during conformational transition in human serum albumin, (ii) the identification of possible routes of metal migration in hemocyanins, and (iii) the prediction of mutations to generate convenient metal-binding sites for de novo biocatalysts. This study shows that BioMetAll offers a versatile platform for numerous fields of research at the interface between inorganic chemistry and biology and allows to highlight the role of the preorganization of the protein backbone as a marker for metal binding. BioMetAll is an open-source application available at https://github.com/insilichem/biometall.