Polymorphism Pro64His within galectin-3 has functional consequences at proteome level in thyroid cells.

Introduction: The single nucleotide polymorphism (SNP) rs4644 at codon 64 of galectin-3 (gal-3, gene name: LGALS3), specifying the variant proline (P64) to histidine (H64), is known to affect the protein's functions and has been associated with the risk of several types of cancer, including differentiated thyroid carcinoma (DTC). Materials and methods: To deepen our understanding of the biological effects of this SNP, we analyzed the proteome of two isogenic cell lines (NC-P64 vs. NA-H64) derived from the immortalized non-malignant thyrocyte cell line Nthy-Ori, generated through the CRISPR-Cas9 technique to differ by rs4644 genotype. We compared the proteome of these cells to detect differentially expressed proteins and studied their proteome in relation to their transcriptome. Results: Firstly, we found, consistently with previous studies, that gal-3-H64 could be detected as a monomer, homodimer, and heterodimer composed of one cleaved and one uncleaved monomer, whereas gal-3-P64 could be found only as a monomer or uncleaved homodimer. Moreover, results indicate that rs4644 influences the expression of several proteins, predominantly upregulated in NA-H64 cells. Overall, the differential protein expression could be attributed to the altered mRNA expression, suggesting that rs4644 shapes the function of gal-3 as a transcriptional co-regulator. However, this SNP also appeared to affect post-transcriptional regulatory mechanisms for proteins whose expression was oppositely regulated compared to mRNA expression. It is conceivable that the rs4644-dependent activities of gal-3 could be ascribed to the different modalities of self-dimerization. Conclusion: Our study provided further evidence that rs4644 could affect the gal-3 functions through several routes, which could be at the base of differential susceptibility to diseases, as reported in case-control association studies.

Health related quality of life, physical function, and cognitive performance in mechanically ventilated COVID-19 patients: A long term follow-up study.

BACKGROUND: Survivors of severe COVID-19 related respiratory failure may experience durable functional impairments. We aimed at investigating health-related quality of life (HR-QoL), physical functioning, fatigue, and cognitive outcomes in COVID-19 patients who received invasive mechanical ventilation (IMV). METHODS: Case-series, prospective, observational cohort study at 18 months from hospital discharge. Patients referring to the Intensive Care Unit (ICU) of Humanitas Research Hospital (Milan, Italy) were recruited if they needed IMV due to COVID-19 related respiratory failure. After 18 months, these patients underwent the 6-min walking test (6MWT), the Italian version of the 5-level EQ-5D questionnaire (EQ-5D-5L), the Functional Assessment of Chronic Illness Therapy - Fatigue questionnaire (FACIT-F), the Trail Making Test-B (TMT-B) and the Montreal Cognitive Assessment-BLIND test (MoCA-BLIND). RESULTS: 105 patients were studied. The population's age was 60 ± 10 years on average, with a median Frailty Scale of 2 (Hodgson et al., 2017; Carenzo et al., 2021a [2,3]). EQ-VAS was 80 [70-90] out of 100, walked distance was 406 [331-465] meters, corresponding to about 74 ± 19,1% of the predicted value. FACIT-F score was 43 [36-49] out of 52, and MoCa-BLIND score was 19 (DeSalvo et al., 2006; von Elm et al., 2008; Herdman et al., 2011; Scalone et al., 2015 [16-20]) out of 22. The median TMT-B time was 90 [62-120] seconds. We found a possible age and gender specific effect on HR-QoL and fatigue. CONCLUSIONS: After 18 months from ICU discharge, survivors of severe COVID-19 respiratory failure experience a moderate reduction in HR-QoL, and a severe reduction in physical functioning. Fatigue prevalence is higher in younger patients and in females. Finally, cognitive impairment was present at a low frequency.

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03878-4.

Right Ventricle Response to Major Lung Resection (the RIVER Study).

Backgrounds: Major lung resection is associated with high postoperative morbidity and mortality, especially due to cardiorespiratory complications. Right ventricle (RV) ejection, pulmonary artery (PA) pressure, and tone are tightly coupled. Since the RV is exquisitely sensitive to changes in afterload, an acute increase in RV outflow resistance (i.e., acute pulmonary embolism [PE]) will cause acute RV dilatation and, a reduction of left ventricle compliance too, rapidly spiraling to acute cardiogenic shock and death. We investigated the changing in RV performance after major lung resection. Materials and Methods: We carried out transthoracic echocardiography (TTE) aiming at searching for the incidence of early RV systolic dysfunction (defined as tricuspid annulus plane systolic excursion [TAPSE] <17 cm, S'-tissue Doppler imaging <10 cm/s) and estimate the RV-PA coupling by the TAPSE/pulmonary artery pressures (PAPs) ratio after major lung resection. The TTE has been performed before and immediately after surgery. Results: After the end of the operation the echocardiographic parameters of the RV function worsened. TAPSE decreased from 24 (21 ÷ 28) to 18 (16 ÷ 22) mm (P = 0.015) and PAPs increased from 26 (25 ÷ 30) to 30 (25 ÷ 39) mmHg (P = 0.013). TAPSE/PAPs ratio decreased from 0.85 (0.80 ÷ 0.90) to 0.64 (0.54 ÷ 0.79) mm/mmHg (P = 0.002). Conclusions: In line with previous reports, after major lung resection the increase in afterload reduces the RV function, but the impairment remains clinically not relevant. The different clinical picture of an acute cor pulmonale due to PE implies that the pathogenesis of cardiac failure involves more pathways than the mere mechanic occlusion of the blood flow.

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.

REVersal of nEuromusculAr bLocking Agents in Patients Undergoing General Anaesthesia (REVEAL Study).

Background: Neuromuscular blocking agent (NMBA) monitoring and reversals are key to avoiding residual curarization and improving patient outcomes. Sugammadex is a NMBA reversal with favorable pharmacological properties. There is a lack of real-world data detailing how the diffusion of sugammadex affects anesthetic monitoring and practice. Methods: We conducted an electronic health record analysis study, including all adult surgical patients undergoing general anesthesia with orotracheal intubation, from January 2016 to December 2019, to describe changes and temporal trends of NMBAs and NMBA reversals administration. Results: From an initial population of 115,046 surgeries, we included 37,882 procedures, with 24,583 (64.9%) treated with spontaneous recovery from neuromuscular block and 13,299 (35.1%) with NMBA reversals. NMBA reversals use doubled over 4 years from 25.5% to 42.5%, mainly driven by sugammadex use, which increased from 17.8% to 38.3%. Rocuronium increased from 58.6% (2016) to 94.5% (2019). Factors associated with NMBA reversal use in the multivariable analysis were severe obesity (OR 3.33 for class II and OR 11.4 for class III obesity, p-value < 0.001), and high ASA score (OR 1.47 for ASA III). Among comorbidities, OSAS, asthma, and other respiratory diseases showed the strongest association with NMBA reversal administration. Conclusions: Unrestricted availability of sugammadex led to a considerable increase in pharmacological NMBA reversal, with rocuronium use also rising. More research is needed to determine how unrestricted and safer NMBA reversal affects anesthesia intraoperative monitoring and practice.

Kinetics of the Cell Cycle Arrest Biomarkers (TIMP2 and IGFBP7) for the Diagnosis of Acute Kidney Injury in Critically Ill COVID-19 Patients.

BACKGROUND: Acute kidney injury (AKI) is highly prevalent in critical COVID-19 patients. The diagnosis and staging of AKI are based on serum creatinine (sCr) and urinary output criteria, with limitations in the functional markers. New cell-cycle arrest biomarkers [TIMP2]*[IGFBP7] have been proposed for early detection of AKI, but their role in critically ill COVID-19 patients is poorly understood. METHODS: We conducted an observational study to assess the performance of [TIMP2]*[IGFBP7] for the detection of AKI in critical COVID-19 patients admitted to our intensive care unit (ICU). We sampled urinary [TIMP2]*[IGFBP7] levels at ICU admission, 12 h, 24 h, and 48 h, and compared the results to the development of AKI, as well as baseline and laboratory data. RESULTS: Forty-one patients were enrolled. The median age was 66 years [57-72] and most were males (85%). Thirteen patients (31.7%) developed no/mild stage AKI, 19 patients (46.3%) moderate AKI, and nine patients (22.0%) severe AKI. The ICU mortality was 29.3%. sCr levels in the Emergency Department or at ICU admission were not significantly different according to AKI stage. [TIMP-2]*[IGFBP-7] urinary levels were elevated in severe AKI at 12 h after ICU admission, but not at ICU admission or 24 h or 48 h after ICU admission. CONCLUSION: Urinary biomarkers [TIMP-2]*[IGFBP-7] were generally increased in this population with a high prevalence of AKI, and were higher in patients with severe AKI measured at 12 h from ICU admission. Further studies are needed to evaluate the best timing of these biomarkers in this population.

Short-term health-related quality of life, physical function and psychological consequences of severe COVID-19.

BACKGROUND: Survivors of severe COVID-19 are at risk of impaired health-related quality of life (HRQoL) and persistent physical and psychological disability after ICU and hospital discharge. The subsequent social burden is a major concern. We aimed to assess the short-term HRQoL, physical function and prevalence of post-traumatic stress symptoms of invasively mechanically ventilated COVID-19 patients treated in our ICU. METHODS: Prospective, observational cohort study in a follow-up clinic. Patients completed a 6-min walking test (6MWT) to assess their cardio-pulmonary function around 2 months (early follow-up) from hospital discharge, the EQ-5D-5L questionnaire for quality of life assessment around 2 months and at 6 months from hospital discharge and an anonymous web-based Impact of Event Scale-Revised (IES-R) questionnaire for Post-Traumatic Stress symptoms at 2 months. RESULTS: 47 patients attended our follow-up program, mean age 59 ± 10 years, median pre-morbid Clinical Frailty Scale (CFS) 2 [2-3]. The median distance walked in 6 min was 470 [406-516] m, 83 [67-99]% of the predicted value. Overall 1 out 3 patients and 4/18 (22%) among those with a good functional baseline prior to COVID-19 (CFS of 1 or 2) had lower (84%) than predicted 6MWT. EQ-5D-5L quality of life VAS was 80 [70-90] out of 100 at early follow-up with a slight improvement to 85 [77.5-90] at 6 months. Mobility, self-care and usual activities improved between the two timepoints, while pain/discomfort and depression/anxiety did not improve or got worse. The IES-R total score was greater than the threshold for concern of 1.6 in 27/41(66%) respondents. CONCLUSIONS: Patients recovering from severe COVID-19 requiring invasive mechanical ventilation surviving hospital discharge present with early mild to moderate functional impairment, mildly reduced quality of life from hospital discharge with an overall improvement of mobility, self-care and the ability of performing usual activities, while a worsening of pain and depression/anxiety symptoms at 6 months and a large proportion of symptoms of post-traumatic distress soon after hospital discharge.

Pro64His (rs4644) Polymorphism Within Galectin-3 Is a Risk Factor of Differentiated Thyroid Carcinoma and Affects the Transcriptome of Thyrocytes Engineered via CRISPR/Cas9 System.

Background: Galectin-3 (LGALS3) is an important glycoprotein involved in the malignant transformation of thyrocytes acting in the extracellular matrix, cytoplasm, and nucleus where it regulates TTF-1 and TCF4 transcription factors. Within LGALS3 gene, a common single-nucleotide polymorphism (SNP) (c.191C>A, p.Pro64His; rs4644) encoding for the variant Proline to Histidine at codon 64 has been extensively studied. However, data on rs4644 in the context of thyroid cancer are lacking. Thus, the aim of the present work was to evaluate the role of the rs4644 SNP as risk factor for differentiated thyroid cancer (DTC) and to determine the effect on the transcriptome in thyrocytes. Methods: A case/control association study in 1223 controls and 1142 unrelated consecutive DTC patients was carried out to evaluate the association between rs4644-P64H and the risk of DTC. We used the nonmalignant cell line Nthy-Ori (rs4644-C/A) and the CRISPR/Cas9 technique to generate isogenic cells carrying either the rs4644-A/A or rs4644-C/C homozygosis. Then, the transcriptome of the derivative and unmodified parental cells was analyzed by RNA-seq. Genes differentially expressed were validated by quantitative reverse transcription PCR and further tested in the parental Nthy-Ori cells after LGALS3 gene silencing, to investigate whether the expression of target genes was dependent on galectin-3 levels. Results: rs4644 AA genotype was associated with a reduced risk of DTC (compared with CC, ORadj = 0.66; 95% confidence interval = 0.46-0.93; Pass = 0.02). We found that rs4644 affects galectin-3 as a transcriptional coregulator. Among 34 genes affected by rs4644, HES1, HSPA6, SPC24, and NHS were of particular interest since their expression was rs4644-dependent (CC>AA for the first and AA>CC for the others), also in 574 thyroid tissues of Genotype-Tissue Expression (GTEx) biobank. Moreover, the expression of these genes was regulated by LGALS3-silencing. Using the proximity ligation assay in Nthy-Ori cells, we found that the TTF-1 interaction was genotype dependent. Conclusions: Our data show that in thyroid, rs4644 is a trans-expression quantitative trait locus that can modify the transcriptional expression of downstream genes, through the modulation of TTF-1.

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.