mTert induction in p21-positive cells counteracts capillary rarefaction and pulmonary emphysema.

Lung diseases develop when telomeres shorten beyond a critical point. We constructed a mouse model in which the catalytic subunit of telomerase (mTert), or its catalytically inactive form (mTertCI), is expressed from the p21Cdkn1a locus. Expression of either TERT or TERTCI reduces global p21 levels in the lungs of aged mice, highlighting TERT non-canonical function. However, only TERT reduces accumulation of very short telomeres, oxidative damage, endothelial cell (ECs) senescence and senile emphysema in aged mice. Single-cell analysis of the lung reveals that p21 (and hence TERT) is expressed mainly in the capillary ECs. We report that a fraction of capillary ECs marked by CD34 and endowed with proliferative capacity declines drastically with age, and this is counteracted by TERT but not TERTCI. Consistently, only TERT counteracts decline of capillary density. Natural aging effects are confirmed using the experimental model of emphysema induced by VEGFR2 inhibition and chronic hypoxia. We conclude that catalytically active TERT prevents exhaustion of the putative CD34 + EC progenitors with age, thus protecting against capillary vessel loss and pulmonary emphysema.

Efficient generation of embryonic stem cells from single blastomeres of cryopreserved mouse embryos in the presence of signalling modulators.

CONTEXT: Derivation of embryonic stem cells (ESC) from single blastomeres is an interesting alternative to the use of whole blastocysts, but derivation rates are lower and the requirements for successful ESC obtention are still poorly defined. AIMS: To investigate the effects of embryo cryopreservation and of signalling modulators present during embryo culture and/or ESC establishment on ESC derivation efficiency from single 8-cell mouse blastomeres. METHOD: Fresh and cryopreserved 2-cell embryos were cultured and biopsied at the 8-cell stage. Single blastomeres were cultured in the presence of 2i or R2i cocktails, with or without adrenocorticotropic hormone (ACTH). We analysed ESC derivation efficiencies and characterised pluripotency genes expression and karyotype integrity of the resulting lines. We also evaluated the impact of embryo preculture with R2i on epiblast cell numbers and derivation rates. KEY RESULTS: The ESC generation was not compromised by embryo cryopreservation and ACTH was dispensable under most of the conditions tested. While 2i and R2i were similarly effective for ESC derivation, R2i provided higher karyotype integrity. Embryo preculture with R2i yielded increased numbers of epiblast cells but did not lead to increased ESC generation. CONCLUSIONS: Our findings help to define a simplified and efficient procedure for the establishment of mouse ESC from single 8-cell blastomeres. IMPLICATIONS: This study will contribute to improving the potential of this experimental procedure, providing a tool to investigate the developmental potential of blastomeres isolated from different embryonic stages and to reduce the number of embryos needed for ESC derivation.

Decellularized Extracellular Matrix-Based Bioinks for Tendon Regeneration in Three-Dimensional Bioprinting.

In the last few years, attempts to improve the regeneration of damaged tendons have been rising due to the growing demand. However, current treatments to restore the original performance of the tissue focus on the usage of grafts; although, actual grafts are deficient because they often cannot provide enough support for tissue regeneration, leading to additional complications. The beneficial effect of combining 3D bioprinting and dECM as a novel bioink biomaterial has recently been described. Tendon dECMs have been obtained by using either chemical, biological, or/and physical treatments. Although decellularization protocols are not yet standardized, recently, different protocols have been published. New therapeutic approaches embrace the use of dECM in bioinks for 3D bioprinting, as it has shown promising results in mimicking the composition and the structure of the tissue. However, major obstacles include the poor structural integrity and slow gelation properties of dECM bioinks. Moreover, printing parameters such as speed and temperature have to be optimized for each dECM bioink. Here, we show that dECM bioink for 3D bioprinting provides a promising approach for tendon regeneration for future clinical applications.

Oncological outcome of wide anatomic resection with partial mesorectal excision in patients with upper and middle rectal cancer.

AIM: The aim was to investigate the influence of distal resection margin and extent of mesorectal excision on long-term oncological outcomes. METHOD: Consecutive patients with upper and middle third rectal cancer from June 2006 to February 2016 were reviewed. Patients were divided into four groups depending on the distal margin considered as a surrogate marker of the extension of mesorectal excision (Q1 ≤10 mm, Q2 11-20 mm, Q3 21-30 mm, Q4 ≥31 mm). Local-recurrence-free survival (LRFS), disease-free survival (DFS) and overall survival (OS) were estimated. Cox regression models were used to investigate the influence of surgical and clinicopathological variables on prognosis by adjusting for confounding factors. RESULTS: Two hundred and eleven patients with mid (125) and upper (86) rectal cancer underwent wide mesorectal excision. The median follow-up was 48.64 months (interquartile range 28-63). 17.5% patients developed recurrence. The 5-year LRFS, DFS and OS for all patients were 93.20%, 83.89% and 80.1%, respectively, with no statistically significant differences between groups (LRFS, P = 0.601; DFS, P = 0.487; OS, P = 0.468). In the multivariable analysis the recurrences and survival were associated with the quality of the mesorectum (LRFS, hazard ratio 10.629, 95% CI 2.324-48.610, P = 0.002; DFS, hazard ratio 2.789, 95% CI 1.314-5.922, P = 0.008). CONCLUSION: A wide anatomical resection with partial mesorectal excision and shorter distal resection margin does not jeopardize the oncological outcomes.

Search for Non-Protein Protease Inhibitors Constituted with an Indole and Acetylene Core.

A possible inhibitor of proteases, which contains an indole core and an aromatic polar acetylene, was designed and synthesized. This indole derivative has a molecular architecture kindred to biologically relevant species and was obtained through five synthetic steps with an overall yield of 37% from the 2,2'-(phenylazanediyl)di(ethan-1-ol). The indole derivative was evaluated through docking assays using the main protease (SARS-CoV-2-Mpro) as a molecular target, which plays a key role in the replication process of this virus. Additionally, the indole derivative was evaluated as an inhibitor of the enzyme kallikrein 5 (KLK5), which is a serine protease that can be considered as an anticancer drug target.

Meaning-making as a mediator of anxiety and depression reduction during cognitive behavioral therapy intervention in participants with adjustment disorders.

There is a consensus among researchers about the link between low meaning in life and anxiety and depressive symptoms. One unanswered question is whether meaning-making is a mediator of the change in anxiety and depression symptoms in participants with adjustment disorders during cognitive behavioural therapy (CBT) treatment. The aims of this study were (a) to analyse whether there was meaning-making during the application of the CBT, (b) to analyse whether meaning-making was a mediator of anxiety psychopathology and (c) to analyse whether meaning-making was a mediator of depressive symptoms. The sample was composed of 115 patients who satisfied the full Diagnostic and Statistical Manual of Mental Disorders (DSM)-5 criteria for adjustment disorder as their primary diagnosis and completed CBT treatment in a primary care mental health service: 74.78% women, n = 86, and 25.22% men, n = 29, with a mean age of 41.89 (standard deviation [SD] = 10.39) years. The diagnosis was established using the Structured Clinical Interview for DSM-5 (SCID-5), and participants filled out the Beck Anxiety Inventory, the Beck Depression Inventory and Purpose in Life questionnaires. The therapists were clinical psychologists with experience in clinical assessment. A repeated-measures analysis of variance (ANOVA) and two mediation analyses using the bootstrap method were performed. The results indicated that (a) There was meaning-making during the CBT because the treated sample showed a statistically significant improvement in meaning in life, and (b) meaning-making during the CBT was a partial mediator between anxiety symptoms and depressive symptoms before and after the treatment. The present study suggests that meaning in life could be an important variable in the psychopathology of adjustment disorders.

Wnt pathway modulation generates blastomere-derived mouse embryonic stem cells with different pluripotency features.

PURPOSE: This study aimed to determine the role of Wnt pathway in mouse embryonic stem cell (mESC) derivation from single blastomeres isolated from eight-cell embryos and in the pluripotency features of the mESC established. METHODS: Wnt activator CHIR99021, Wnt inhibitor IWR-1-endo, and MEK inhibitor PD0325901 were used alone or in combination during ESC derivation and maintenance from single blastomeres biopsied from eight-cell embryos. Alkaline phosphatase activity, FGF5 levels, expression of key pluripotency genes, and chimera formation were assessed to determine the pluripotency state of the mESC lines. RESULTS: Derivation efficiencies were highest when combining pairs of inhibitors (15-24.7%) than when using single inhibitors or none (1.4-10.1%). Full naïve pluripotency was only achieved in CHIR- and 2i-treated mESC lines, whereas IWR and PD treatments or the absence of treatment resulted in co-existence of naïve-like and primed-like pluripotency features. IWR + CHIR- and IWR + PD-treated mESC displayed features of primed pluripotency, but IWR + CHIR-treated lines were able to generate germline-competent chimeric mice, resembling the predicted properties of formative pluripotency. CONCLUSION: Wnt and MAPK pathways have a key role in the successful derivation and pluripotency features of mESC from single precompaction blastomeres. Modulation of these pathways results in mESC lines with various degrees of naïve-like and primed-like pluripotency features.

Early intervention with cognitive behavioral therapy reduces sick leave duration in people with adjustment, anxiety and depressive disorders.

Background: Early intervention in workers diagnosed with mental disorders is associated with a lower incidence of relapse and shorter sick leave. However, no studies have been carried out on the effect of early intervention using an evidence-based therapy, Cognitive Behavioral Therapy (CBT), on people with sick leave.Aims: The objectives of the present study are to study whether the type of intervention (early or late) will affect the total duration of the sick leave, the partial duration of the sick leave, the duration of the psychotherapy and the time until return to work after the psychotherapy ends. The sample was composed of 167 participants who were on sick leave for adjustment disorders, anxiety disorders or depressive disorder.Results: The participants who had early intervention with CBT had a significantly shorter duration of total sick leave and partial sick leave, and a shorter time until returning to work after the psychotherapy ended than those who had late intervention. There were no statistically differences in the duration or efficacy of the psychotherapy.Conclusion: We can suggest that providing early access to CBT significantly reduces the length of sick leave in patients with mental disorders.

Rational Engineering of Multiple Active Sites in an Ester Hydrolase.

Effects of altering the properties of an active site in an enzymatic homogeneous catalyst have been extensively reported. However, the possibility of increasing the number of such sites, as commonly done in heterogeneous catalytic materials, remains unexplored, particularly because those have to accommodate appropriate residues in specific configurations. This possibility was investigated by using a serine ester hydrolase as the target enzyme. By using the Protein Energy Landscape Exploration software, which maps ligand diffusion and binding, we found a potential binding pocket capable of holding an extra catalytic triad and oxyanion hole contacts. By introducing two mutations, this binding pocket became a catalytic site. Its substrate specificity, substrate preference, and catalytic activity were different from those of the native site of the wild type ester hydrolase and other hydrolases, due to the differences in the active site architecture. Converting the binding pocket into an extra catalytic active site was proven to be a successful approach to create a serine ester hydrolase with two functional reactive groups. Our results illustrate the accuracy and predictive nature of modern modeling techniques, opening novel catalytic opportunities coming from the presence of different catalytic environments in single enzymes.

Reducing the time until psychotherapy initiation reduces sick leave duration in participants diagnosed with anxiety and mood disorders.

BACKGROUND: Sick leave in patients with a mental disorder is characterized by having a long duration. Studies suggest that the time until a patient on sick leave for a common mental health disorder initiates evaluation and treatment by a healthcare professional is an important factor in the duration of the sick leave. However, in these studies, the intervention was not performed by a mental health specialist. The aim of this study was to find out whether the length of sick leave was associated with the time before initiating psychotherapy, age, time until returning to work after psychotherapy ends, and duration of psychotherapy. In a further analysis, we examined whether the model composed of age, duration of psychotherapy, and time before initiating psychotherapy predicted the length of sick leave. METHOD: The sample consisted of 2,423 participants, 64.1% (n = 1,554) women and 35.9% (n = 869) men, who were on sick leave for anxiety disorders or depressive disorder. RESULTS: The total duration of the sick leave of participants diagnosed with depression and anxiety was positively associated with the time before beginning psychotherapy. Time before beginning psychotherapy predicted the length of sick leave when the variables age and duration of psychotherapy were controlled. CONCLUSION: It is necessary to reduce the time until beginning psychotherapy in people on sick leave for common mental disorders.

Endoscopic tattooing of colorectal neoplasms removed by laparoscopy: a proposal for selective marking.

BACKGROUND AND AIM: Preoperative endoscopic tattooing is an effective procedure to identify small intraoperative neoplasms. However, there are no defined criteria with regard to the indications for endoscopic tattooing of these lesions at the time of diagnosis. The aim of this study was to establish endoscopic criteria that allow the selection of patients who will need a tattoo during the diagnostic colonoscopy. METHODS: An ambispective study of patients undergoing laparoscopy due to a colorectal neoplasia who underwent endoscopic tattooing during the period from 2007-2013 and 2016-2017. According to the endoscopic description of the neoplasms, the classification was polypoid lesions, neoplasms occupying < 50% or ≥ 50% of the intestinal lumen and stenosing neoplasias. RESULTS: Tattooing of the lesion was performed in 120 patients and the same lesions were identified during surgery in 114 (95%) cases. Most of the neoplasias described as polypoids and neoplasias that occupied < 50% of the intestinal lumen were not visualized during surgery and therefore required a tattoo (33 of 42 and 18 of 26 respectively, p = 0.0001, X2). On the other hand, stenosing lesions or neoplasias occupying ≥ 50% of the intestinal lumen were mostly identified during surgery (15 of 15 and 36 of 37 respectively, p = 0.0001, X2) without the need for a tattoo. Overall, the identification of neoplasms according to established criteria was 98%. CONCLUSION: These results suggest that it is possible to establish endoscopic criteria that allow a successful selective tattooing during diagnostic endoscopy.

In vitro fungicidal activities of anidulafungin, caspofungin, and micafungin against Candida glabrata, Candida bracarensis, and Candida nivariensis evaluated by time-kill studies.

Anidulafungin, caspofungin, and micafungin killing activities against Candida glabrata, Candida bracarensis, and Candida nivariensis were evaluated by the time-kill methodology. The concentrations assayed were 0.06, 0.125, and 0.5 µg/ml, which are achieved in serum. Anidulafungin and micafungin required between 13 and 26 h to reach the fungicidal endpoint (99.9% killing) against C. glabrata and C. bracarensis. All echinocandins were less active against C. nivariensis.

Postoperative peritoneal infection enhances migration and invasion capacities of tumor cells in vitro: an insight into the association between anastomotic leak and recurrence after surgery for colorectal cancer.

OBJECTIVE: The aim of this study was to investigate the effect of postoperative peritoneal infection on proliferation, migration, and invasion capacities of cancer cells lines in vitro after surgery for colorectal cancer. BACKGROUND: Anastomotic leakage is associated with higher rates of recurrence after surgery for colorectal cancer. However, the mechanisms responsible are unknown. We hypothesized that the infection-induced inflammatory response may enhance tumor progression features of residual cancer cells. METHODS: Prospective matched cohort study. Patients undergoing surgery for colorectal cancer with curative intent (January 2008-March 2012) were included. Patients who had an anastomotic leak or intra-abdominal abscess were included in the infection group (n=47). For each case patient, another patient with an uncomplicated postoperative course was selected for the control group (n=47).In vitro treatments on cancer cell lines (MDA-MB-231 and SW620) were performed using baseline and postoperative serum and peritoneal fluid samples to determine cell proliferation and cell migration/invasion activities. RESULTS: Postoperative peritoneal fluid from infected patients enhanced both cell migration (infection: 140±85 vs control: 94±30; P=0.016) and cell invasion (infection: 117±31 vs control: 103±16; P=0.024) capacities of cancer cell lines. With serum samples, these effects were only observed in cell migration assays (infection: 98±28 vs control: 87±17; P=0.005). Some minor activation of cell proliferation was observed by treatment with serum from infection group. Two-year cumulative disease-free survival was significantly lower in patients with postoperative peritoneal infection (infection: 77.6% vs control: 90.6%; P=0.032). CONCLUSIONS: Our results suggest that postoperative peritoneal infection enhances the invasive capacity of residual tumor cells after surgery, thus facilitating their growth to recurrent tumors.

In vitro determination of the short-chain synthetic peptide RP13 antimicrobial activity.

BACKGROUND: The proliferation of antibiotic-resistant microorganisms, along with the lack of new drugs against them, has elicited the interest of the scientific community on the study and development of endogenous synthetic compounds with bacteriostatic or bactericidal activity. In recent years, several short-chain, low molecular weight peptides isolated from natural sources such as plants and animals have demonstrated an array of antimicrobial activities. Despite having structural characteristics similar to microbicidal peptides isolated from human platelets, peptide RP11 does not exhibit antimicrobial activity. OBJECTIVE: In vitro determination of the antimicrobial activity of the synthetic peptide RP13. MATERIAL AND METHODS: Peptide RP13 was prepared modifying the original amino acids sequence of peptide RP11, reversing the position of the amino acids lysine and tyrosine in order to modify the conformation of the original peptide. These amino acids are localized close to the N-terminus of the peptidic chain. Peptide RP13 was prepared in solution using conventional methods for peptide synthesis. The antimicrobial activity of RP13 was assessed against the microorganisms S. aureus, E. faecalis and E. coli in a test solution and later evaluated by cultivation of plates during the first 2 h after inoculation of bacteria. RP13 activity antimicrobial was compared against tetracycline, a broad-spectrum antibiotic. RESULTS: The new peptide RP13, resulting form the structural modification of the amino acid sequence of peptide RP11, displayed antimicrobial activity. RP13 demonstrated to be more efficient inhibiting the growth of gram-positive than gramnegative bacteria. CONCLUSIONS: The structural modification of peptide RP11, obtained from human platelets, resulted in a new peptide with improved antimicrobial activity. These results clearly demonstrate that peptides of natural origin, as well as their synthetic analogs, represent an attractive alternative against pathogenic agents.

[Treatment of colorectal cancer with unresectable metastasis with chemotherapy without primary tumor resection: analysis of tumor-related complications]. / Tratamiento del cáncer colorrectal con metástasis irresecables con quimioterapia sin resección del primario: análisis de las complicaciones relacionadas con el tumor.

INTRODUCTION: Although the conventional treatment of patients with stage iv colorectal cancer involves resection of the primary tumor followed by chemotherapy, several studies suggest that in patients with few symptoms the first and only treatment should be chemotherapy. The objective of this study is to analyze the complications related to the primary tumor in a series of patients with unresectable metastatic colorectal cancer treated with chemotherapy without surgery. MATERIAL AND METHODS: Retrospective descriptive study. The study included all patients with unresectable metastatic colorectal cancer treated with chemotherapy without resection of the primary tumor (January 2007-February 2011). RESULTS: The mean age of the 61 patients analyzed was 67±13 years and the performance status was 0-1 in 53 (87%). Twenty (33%) patients developed complications during follow-up. The most common complication was intestinal obstruction in 15 (25%) patients followed by perforation. Complications required surgery in 6 (10%) cases. We did not find differences in patient characteristics between those who had a complication and those without, although the complication rate in patients with a colonic stent (53%) was twice that of other patients (26%). CONCLUSIONS: Chemotherapy without surgery is a good option in most patients with unresectable metastatic colorectal cancer. However, although the percentage of patients requiring surgery is low, the total number of complications related to the primary tumor is not negligible. Studies are needed to identify those patients in whom a prophylactic colectomy could be indicated.

Circulating metabolic markers after surgery identify patients at risk for severe postoperative complications: a prospective cohort study in colorectal cancer.

BACKGROUND: Early detection of postoperative complications after colorectal cancer (CRC) surgery is associated with improved outcomes. The aim was to investigate early metabolomics signatures capable to detect patients at risk for severe postoperative complications after CRC surgery. MATERIALS AND METHODS: Prospective cohort study of patients undergoing CRC surgery from 2015 to 2018. Plasma samples were collected before and after surgery, and analyzed by mass spectrometry obtaining 188 metabolites and 21 ratios. Postoperative complications were registered with Clavien-Dindo Classification and Comprehensive Complication Index. RESULTS: One hundred forty-six patients were included. Surgery substantially modified metabolome and metabolic changes after surgery were quantitatively associated with the severity of postoperative complications. The strongest positive relationship with both Clavien-Dindo and Comprehensive Complication Index (ß=4.09 and 63.05, P <0.001) corresponded to kynurenine/tryptophan, against an inverse relationship with lysophosphatidylcholines (LPCs) and phosphatidylcholines (PCs). Patients with LPC18:2/PCa36:2 below the cut-off 0.084 µM/µM resulted in a sevenfold higher risk of major complications (OR=7.38, 95% CI: 2.82-21.25, P <0.001), while kynurenine/tryptophan above 0.067 µM/µM a ninefold (OR=9.35, 95% CI: 3.03-32.66, P <0.001). Hexadecanoylcarnitine below 0.093 µM displayed a 12-fold higher risk of anastomotic leakage-related complications (OR=11.99, 95% CI: 2.62-80.79, P =0.004). CONCLUSION: Surgery-induced phospholipids and amino acid dysregulation is associated with the severity of postoperative complications after CRC surgery, including anastomotic leakage-related outcomes. The authors provide quantitative insight on metabolic markers, measuring vulnerability to postoperative morbidity that might help guide early decision-making and improve surgical outcomes.

A point cloud segmentation framework for image-based spatial transcriptomics.

Recent progress in image-based spatial RNA profiling enables to spatially resolve tens to hundreds of distinct RNA species with high spatial resolution. It presents new avenues for comprehending tissue organization. In this context, the ability to assign detected RNA transcripts to individual cells is crucial for downstream analyses, such as in-situ cell type calling. Yet, accurate cell segmentation can be challenging in tissue data, in particular in the absence of a high-quality membrane marker. To address this issue, we introduce ComSeg, a segmentation algorithm that operates directly on single RNA positions and that does not come with implicit or explicit priors on cell shape. ComSeg is applicable in complex tissues with arbitrary cell shapes. Through comprehensive evaluations on simulated and experimental datasets, we show that ComSeg outperforms existing state-of-the-art methods for in-situ single-cell RNA profiling and in-situ cell type calling. ComSeg is available as a documented and open source pip package at https://github.com/fish-quant/ComSeg .

Comprehensive review of the state-of-the-art in corneal 3D bioprinting, including regulatory aspects.

The global shortage of corneal transplants has spurred an urgency in the quest for efficient treatments. This systematic review not only provides a concise overview of the current landscape of corneal morphology, physiology, diseases, and conventional treatments but crucially delves into the forefront of tissue engineering for corneal regeneration. Emphasizing cellular and acellular components, bioprinting techniques, and pertinent biological assays, it explores optimization strategies for manufacturing and cost-effectiveness. To bridge the gap between research and industrial production, the review outlines the essential regulatory strategy required in Europe, encompassing relevant directives, frameworks, and governing bodies. This comprehensive regulatory framework spans the entire process, from procuring initial components to marketing and subsequent product surveillance. In a pivotal shift towards the future, the review culminates by highlighting the latest advancements in this sector, particularly the integration of tissue therapy with artificial intelligence. This synergy promises substantial optimization of the overall process, paving the way for unprecedented breakthroughs in corneal regeneration. In essence, this review not only elucidates the current state of corneal treatments and tissue engineering but also outlines regulatory pathways and anticipates the transformative impact of artificial intelligence, providing a comprehensive guide for researchers, practitioners, and policymakers in the field.

Single-cell and spatial transcriptomics analysis of non-small cell lung cancer.

Lung cancer is the second most frequently diagnosed cancer and the leading cause of cancer-related mortality worldwide. Tumour ecosystems feature diverse immune cell types. Myeloid cells, in particular, are prevalent and have a well-established role in promoting the disease. In our study, we profile approximately 900,000 cells from 25 treatment-naive patients with adenocarcinoma and squamous-cell carcinoma by single-cell and spatial transcriptomics. We note an inverse relationship between anti-inflammatory macrophages and NK cells/T cells, and with reduced NK cell cytotoxicity within the tumour. While we observe a similar cell type composition in both adenocarcinoma and squamous-cell carcinoma, we detect significant differences in the co-expression of various immune checkpoint inhibitors. Moreover, we reveal evidence of a transcriptional "reprogramming" of macrophages in tumours, shifting them towards cholesterol export and adopting a foetal-like transcriptional signature which promotes iron efflux. Our multi-omic resource offers a high-resolution molecular map of tumour-associated macrophages, enhancing our understanding of their role within the tumour microenvironment.