Wearable Health Technology for Preoperative Risk Assessment in Elderly Patients: The WELCOME Study

Preoperative identification of high-risk groups has been extensively studied to improve patients’ outcomes. Wearable devices, which can track heart rate and physical activity data, are starting to be evaluated for patients’ management. We hypothesized that commercial wearable devices (WD) may provide data associated with preoperative evaluation scales and tests, to identify patients with poor functional capacity at increased risk for complications. We conducted a prospective observational study including seventy-year-old patients undergoing two-hour surgeries under general anesthesia. Patients were asked to wear a WD for 7 days before surgery. WD data were compared to preoperatory clinical evaluation scales and with a 6-min walking test (6MWT). We enrolled 31 patients, with a mean age of 76.1 (SD ± 4.9) years. There were 11 (35%) ASA 3–4 patients. 6MWT results averaged 328.9 (SD ± 99.5) m. Daily steps and 2 as recorded using WD and were associated with 6MWT performance (R = 0.56, p = 0.001 and r = 0.58, p = 0.006, respectively) and clinical evaluation scales. This is the first study to evaluate WD as preoperative evaluation tools; we found a strong association between 6MWT, preoperative scales, and WD data. Low-cost wearable devices are a promising tool for the evaluation of cardiopulmonary fitness. Further research is needed to validate WD in this setting

Strategies for single base gene editing in an immortalized human cell line by CRISPR/Cas9 technology

The use of CRISPR/Cas9 system has rapidly grown in the last years. Here, the optimization of gene editing of a single-nucleotide polymorphism in a human non-malignant somatic cell line of thyrocytes (Nthy-Ori) was described highlighting strategies for overcoming the problems concerning the delivery and off-targets. We employed both lentivirus and chemical lipids as delivery agents and two strategies for creating the double-strand breaks (DSB). The former induced a DSB by a classical Cas9 nuclease (standard strategy), while the second one employed a modified Cas9 creating a single-strand break (SSB). The knock-in was carried out using a single-stranded donor oligonucleotide or the HR410-PA donor vector (HR). The desired cells could be obtained by combining the double nickase system with the HR vector transfected chemically. This result could be due to the type of DSB, likely processed mainly by non-homologous end joining when blunt (standard strategy) and by HR when overhanging (double nickase). Our results showed that the double nickase is suitable for knocking-in the immortalized Nthy-Ori cell line, while the standard CRISPR/Cas9 system is suitable for gene knock-out creating in/del mutations

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

"Valutazione delle migliori strategie basate sulla tecnologia CRISPR/Cas9 per effettuare knock-in genico dello SNP rs4644 del gene LGALS3"

Il sistema Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) è un meccanismo di difesa immuno-adattativo utilizzato da Archea e Bacteria per la degradazione di materiale genetico estraneo. Sono stati individuati tre diversi sottotipi di sistemi (Tipo I, II e III) che differiscono per il numero e per la tipologia di endonucleasi coinvolte. L’interesse della comunità scientifica si è rivolto in particolar modo verso il sistema CRISPR/Cas9 di tipo II, individuato per la prima volta in Streptococcus pyogenes, che risulta essere il migliore e il più utilizzato metodo per effettuare manipolazioni geniche, come ad esempio knock-in, knock-out e knock-down. Infatti, negli ultimi anni, grazie alla sua semplicità e versatilità, rispetto ad altre tecnologie in grado di indurre modificazioni genetiche (TALEN, ZFN), questa metodica è stata impiegata in diversi ambiti, quali terapia genica, ricerca di nuovi farmaci e biologia sintetica. Il CRISPR/Cas9 di tipo II è costituito da una ribonucleoproteina, data dall’endonucleasi Cas9 a cui fa seguito un RNA guida di circa 20-nt (sgRNA), che dirige l’endonucleasi stessa. La ribonucleoproteina è in grado di identificare la sequenza trinucleotidica (NGG) PAM (Protospacer Adjacent Motifs), presente in più copie a livello del genoma, preceduta da una sequenza complementare al sgRNA, inducendo la formazione di un taglio al doppio filamento di DNA (DSB). La cellula risponde a quest’ultimo con due diversi meccanismi di riparazione: la ricombinazione non omologa (NHEJ), meccanismo error prone che induce mutazioni di tipo inserzione/delezione (indel), e la ricombinazione omologa (HDR), che ripara il danno DSB utilizzando un filamento di DNA stampo, senza inserire mutazioni. Il presente elaborato si inserisce in un ampio progetto di ricerca, il cui scopo è quello di delineare il ruolo funzionale del polimorfismo a singolo nucleotide (SNP) rs4644 nel gene LGALS3, per il quale è stata dimostrata un’associazione con il rischio di sviluppare diversi tipi di cancro. A tal proposito, linee cellulari umane isogeniche che differiscono dalla linea parentale per il solo SNP di interesse saranno create mediante il sistema CRISPR/Cas9. Nello specifico sono stati utilizzati tre approcci differenti. Nel primo caso abbiamo impiegati due vettori, PX458 e PX459, dissimili esclusivamente per il marker di selezione, contenenti il gene codificante per la Cas9, la quale va ad indurre un taglio a livello del genoma inducendo un DSB nel DNA. Nel secondo caso è stata utilizzata una strategia diversa, nota come CRISPR double nickase, che va ad aumentare la specificità, diminuendo il numero di off-target e mantenendo un alto livello di efficienza. In quest’ultimo caso due plasmidi, ciascuno codificante per una Cas9 mutante, sono diretti verso una specifica regione del DNA genomico, dall'RNA guida target specifico. Ciascun complesso Cas9/sgRNA crea un taglio al singolo filamento (SSB) e il doppio nick, creato dai due complessi, mima un DSB. La terza tecnica prevede l’impego di vettori lentivirali come mezzo di trasfezione di un plasmide contenente la Cas9 inducibile da doxyciclina e resistente alla puromicina e di un vettore con sgRNA target e resistenza alla blasticidina. La riparazione del DSB viene mediata da due elementi: un single-stranded donor oligonucleotides (ssODN), avente regioni fiancheggianti omologhe alla regione target e l’allele di interesse posto al centro della sequenza, o un donor vector avente braccia di omologia fiancheggianti il sito interessato dal danno. Lo scopo della presente tesi è quello di individuare non solo il miglior sistema per effettuare il knock-in genico, inteso come l’impiego del CRISPR-cas9 in associazione con il ssODN o in associazione con il donor vector, ma anche la migliore sgRNA. Per quest’ultimo aspetto, ci si è avvalsi dell’utilizzo di un tool bioinormatico, quale CHOPCHOP. Nello specifico, sulla base degli off-target e della qualità sgRNA, sono state selezionate due guide, che riconoscono lo SNP di nostro interesse. I dati preliminari mostrano, complessivamente, una buona specificità ed efficienza da parte delle sgRNAs utilizzate e dei tre approcci adoperati. Ulteriori saggi dovranno essere condotti allo scopo di determinare il miglior metodo e la miglior guida che ci permettano di ottenere le linee cellulari modificate. Analisi in silico come TIDER assay, ma anche genotipizzazione delle linee modificate dovranno essere effettuate per verificare l’effettivo gene-editing

T-type calcium channels drive the proliferation of androgen-receptor negative prostate cancer cells

Background: Androgen deprivation therapy (ADT) is the treatment of choice for metastatic prostate cancer (PCa). After an initial response to ADT, PCa cells can generate castration resistant (CRPC) or neuroendocrine (NEPC) malignancies, which are incurable. T-type calcium channels (TTCCs) are emerging as promising therapeutic targets for several cancers, but their role in PCa progression has never been investigated. Methods: To examine the role of TTCCs in PCa, we analyzed their expression level, copy number variants (CNV) and prognostic significance using clinical datasets (Oncomine and cBioPortal). We then evaluated TTCC expression in a panel of PCa cell lines and measured the effect of their inhibition on cell proliferation and survival using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and caspase assays. Results: TTCCs were upregulated in PCas harboring androgen receptor (AR) mutations; CNV rate was positively associated with PCa progression. Higher expression of one TTCC isoform (CACNA1G) predicted poorer postoperative prognosis in early stage PCa samples. Pharmacological or small interfering RNA (siRNA)-based inhibition of TTCCs caused a decrease in PC-3 cell survival and proliferation. Conclusions: Our results show that TTCCs are overexpressed in advanced forms of PCa and correlate with a poorer prognosis. TTCC inhibition reduces cell proliferation and survival, suggesting that there may be possible value in the therapeutic targeting of TTCCs in advanced PCa

Proteomic analysis of thyrocytes carrying H64 (rs4644) homo-zygotes polymorphism within galectin-3 unveils a carcinogenic profile

Genomic wide association studies and intensive bioinformatic searches of databases have identified several loci associated with genetic predisposition to thyroid cancer [1,2]. Among these, the LGALS3 gene, which encodes galectin-3 protein (gal-3), is a potential candidate to be considered. Within LGALS3 gene, a common single nucleotide polymorphism (SNP) encoding for the variant Proline (P64) to Histidine (H64) at codon 64 (rs4644) has been found to affect function of galectin-3. The aim of this work was to determine proteomes of rs4644 SNP variants in human thyroid cell line (Nthy-Ori). Nthy-Ori cells have a heterozygote genotype C/A allowing the conversion into homozygote genotypes (either A/A and C/C) using the CRISPR/Cas9 gene editing system, as described by Corrado et al. [3]. Gene edited cells were named ORI-CC and ORI-AA, also called gal-3P64 and gal-3H64, respectively. Proteomic analysis of genetic variants of Nthy-Ori cell was performed using two-dimensional gel electrophoresis coupled to mass spectrometry. A total of about 1300 spots were detected on gels from different samples. Then, after com-putational comparison of images, 37 protein spots were found to be differentially expressed in ORI-AA with comparison to ORI-CC cells. In order to identify the proteins potentially correlated with rs4644 mutation, differentially expressed spots were cut from gels, trypsinized and analyzed by nano-LC ESI MS/MS. Ingenuity pathways analysis (IPA) was performed to recognize molecular and cellular functions correlated to differentially expressed proteins. Some of the identified proteins found differentially expressed in gal-3H64 respect to gal3P64 variants, belong to chaperones class as HSPA6, HSPAB1 and to actin-binding protein class, as the calponin 3. IPA analysis suggested the involvement of cancer-related upstream regulators such as TP53, MYCN, and HIF1A1. For the first time, rs4644 SNP variants in thyroid cell were disclosed by a proteomic analysis approach to cause cancer-related molecular changes in non-malignant thyroid cells. [1] Jendrzejewski, J.P.; Sworczak, K.; Comiskey, D.F.; de la Chapelle A. Endokrynol Pol. (2019), 70, 423- 429. [2] Saenko, V.A.; Rogounovitch, T.I. Endocrinol Metab., (2018), 33,164-174. [3] Corrado, A.; Aceto, R.; Silvestri, R.; Dell'Anno, I.; Ricci, B.; Miglietta, S.; Romei, C.; Giovannoni, R.; Poliseno, L.; Evangelista, M.; Vitiello, M.; Cipollini ,M.; Garritano ,S.; Giusti ,L.; Zallocco, L.; Elisei ,R.; Landi, S.; Gemignani, F. Thyroid. , (2021), doi: 10.1089/thy.2020.036

Proteomic analysis of thyrocytes carrying H64 (rs4644) homo-zygotes polymorphism within galectin-3 unveils a carcinogenic profile

Genomic wide association studies and intensive bioinformatic searches of databases have identified several loci associated with genetic predisposition to thyroid cancer [1,2]. Among these, the LGALS3 gene, which encodes galectin-3 protein (gal-3), is a potential candidate to be considered. Within LGALS3 gene, a common single nucleotide polymorphism (SNP) encoding for the variant Proline (P64) to Histidine (H64) at codon 64 (rs4644) has been found to affect function of galectin-3. The aim of this work was to determine proteomes of rs4644 SNP variants in human thyroid cell line (Nthy-Ori). Nthy-Ori cells have a heterozygote genotype C/A allowing the conversion into homozygote genotypes (either A/A and C/C) using the CRISPR/Cas9 gene editing system, as described by Corrado et al. [3]. Gene edited cells were named ORI-CC and ORI-AA, also called gal-3P64 and gal-3H64, respectively. Proteomic analysis of genetic variants of Nthy-Ori cell was performed using two-dimensional gel electrophoresis coupled to mass spectrometry. A total of about 1300 spots were detected on gels from different samples. Then, after com-putational comparison of images, 37 protein spots were found to be differentially expressed in ORI-AA with comparison to ORI-CC cells. In order to identify the proteins potentially correlated with rs4644 mutation, differentially expressed spots were cut from gels, trypsinized and analyzed by nano-LC ESI MS/MS. Ingenuity pathways analysis (IPA) was performed to recognize molecular and cellular functions correlated to differentially expressed proteins. Some of the identified proteins found differentially expressed in gal-3H64 respect to gal3P64 variants, belong to chaperones class as HSPA6, HSPAB1 and to actin-binding protein class, as the calponin 3. IPA analysis suggested the involvement of cancer-related upstream regulators such as TP53, MYCN, and HIF1A1. For the first time, rs4644 SNP variants in thyroid cell were disclosed by a proteomic analysis approach to cause cancer-related molecular changes in non-malignant thyroid cells. [1] Jendrzejewski, J.P.; Sworczak, K.; Comiskey, D.F.; de la Chapelle A. Endokrynol Pol. (2019), 70, 423- 429. [2] Saenko, V.A.; Rogounovitch, T.I. Endocrinol Metab., (2018), 33,164-174. [3] Corrado, A.; Aceto, R.; Silvestri, R.; Dell'Anno, I.; Ricci, B.; Miglietta, S.; Romei, C.; Giovannoni, R.; Poliseno, L.; Evangelista, M.; Vitiello, M.; Cipollini ,M.; Garritano ,S.; Giusti ,L.; Zallocco, L.; Elisei ,R.; Landi, S.; Gemignani, F. Thyroid. , (2021), doi: 10.1089/thy.2020.036

Wearable Health Technology for Preoperative Risk Assessment in Elderly Patients: The WELCOME Study

Preoperative identification of high-risk groups has been extensively studied to improve patients’ outcomes. Wearable devices, which can track heart rate and physical activity data, are starting to be evaluated for patients’ management. We hypothesized that commercial wearable devices (WD) may provide data associated with preoperative evaluation scales and tests, to identify patients with poor functional capacity at increased risk for complications. We conducted a prospective observational study including seventy-year-old patients undergoing two-hour surgeries under general anesthesia. Patients were asked to wear a WD for 7 days before surgery. WD data were compared to preoperatory clinical evaluation scales and with a 6-min walking test (6MWT). We enrolled 31 patients, with a mean age of 76.1 (SD ± 4.9) years. There were 11 (35%) ASA 3–4 patients. 6MWT results averaged 328.9 (SD ± 99.5) m. Daily steps and 𝑉̇𝑂2𝑚𝑎𝑥 as recorded using WD and were associated with 6MWT performance (R = 0.56, p = 0.001 and r = 0.58, p = 0.006, respectively) and clinical evaluation scales. This is the first study to evaluate WD as preoperative evaluation tools; we found a strong association between 6MWT, preoperative scales, and WD data. Low-cost wearable devices are a promising tool for the evaluation of cardiopulmonary fitness. Further research is needed to validate WD in this setting

Wearable Health Technology for Preoperative Risk Assessment in Elderly Patients: The WELCOME Study

Preoperative identification of high-risk groups has been extensively studied to improve patients’ outcomes. Wearable devices, which can track heart rate and physical activity data, are starting to be evaluated for patients’ management. We hypothesized that commercial wearable devices (WD) may provide data associated with preoperative evaluation scales and tests, to identify patients with poor functional capacity at increased risk for complications. We conducted a prospective observational study including seventy-year-old patients undergoing two-hour surgeries under general anesthesia. Patients were asked to wear a WD for 7 days before surgery. WD data were compared to preoperatory clinical evaluation scales and with a 6-min walking test (6MWT). We enrolled 31 patients, with a mean age of 76.1 (SD ± 4.9) years. There were 11 (35%) ASA 3–4 patients. 6MWT results averaged 328.9 (SD ± 99.5) m. Daily steps and 𝑉𝑂2𝑚𝑎𝑥 as recorded using WD and were associated with 6MWT performance (R = 0.56, p = 0.001 and r = 0.58, p = 0.006, respectively) and clinical evaluation scales. This is the first study to evaluate WD as preoperative evaluation tools; we found a strong association between 6MWT, preoperative scales, and WD data. Low-cost wearable devices are a promising tool for the evaluation of cardiopulmonary fitness. Further research is needed to validate WD in this setting.</jats:p

Machine Learning for Early Outcome Prediction in Septic Patients in the Emergency Department

Background: Sepsis is one of the major causes of in-hospital death, and is frequent in patients presenting to the emergency department (ED). Early identification of high-risk septic patients is critical. Machine learning (ML) techniques have been proposed for identification and prognostication of ED septic patients, but these models often lack pre-hospital data and lack validation against early sepsis identification scores (such as qSOFA) and scores for critically ill patients (SOFA, APACHE II). Methods We conducted an electronic health record (EHR) study to test whether interpretable and scalable ML models predict mortality in septic ED patients and compared their performance with clinical scores. Consecutive adult septic patients admitted to ED over 18 months were included. We built ML models, ranging from a simple-classifier model, to unbalanced and balanced logistic regression, and random forest, and compared their performance to qSOFA, SOFA, and APACHE II scores. Results: We included 425 sepsis patients after screening 38,500 EHR for sepsis criteria. Overall mortality was 15.2% and peaked in patients coming from retirement homes (38%). Random forest, like balanced (0.811) and unbalanced logistic regression (0.863), identified patients at risk of mortality (0.813). All ML models outperformed qSOFA, APACHE II, and SOFA scores. Age, mean arterial pressure, and serum sodium were major mortality predictors. Conclusions: We confirmed that random forest models outperform previous models, including qSOFA, SOFA, and APACHE II, in identifying septic patients at higher mortality risk, while maintaining good interpretability. Machine learning models may gain further adoption in the future with increasing diffusion and granularity of EHR data, yielding the advantage of increased scalability compared to standard statistical techniques