In pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid activation. The identity of transcription factors (TFs) that specifically repress ...pro-differentiation genes remains obscure. By targeting ∼1,700 TFs with CRISPR loss-of-function screen, we found that ZBTB11 and ZFP131 are required for embryonic stem cell (ESC) pluripotency. ESCs without ZBTB11 or ZFP131 lose colony morphology, reduce proliferation rate, and upregulate transcription of genes associated with three germ layers. ZBTB11 and ZFP131 bind proximally to pro-differentiation genes. ZBTB11 or ZFP131 loss leads to an increase in H3K4me3, negative elongation factor (NELF) complex release, and concomitant transcription at associated genes. Together, our results suggest that ZBTB11 and ZFP131 maintain pluripotency by preventing premature expression of pro-differentiation genes and present a generalizable framework to maintain cellular potency.
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•A CRISPR screen identifies ZBTB11 and ZFP131 as required TFs for pluripotency•ZBTB11 and ZFP131 bind proximally to pro-differentiation genes•Zbtb11 or Zfp131 mutant ESCs express genes associated with three germ layers•As a result, Zbtb11 or Zfp131 mutant cells lose pluripotency and differentiate
With a highly sensitive CRISPR loss-of-function screen targeting all TFs in the mouse and human genome, Garipler et al. show that ZBTB11 and ZFP131 TFs are required for embryonic stem cell pluripotency. ZBTB11 and ZFP131 maintain poised pro-differentiation genes in a repressed state to preserve pluripotency.
The development of dendritic cells (DCs), including antigen-presenting conventional DCs (cDCs) and cytokine-producing plasmacytoid DCs (pDCs), is controlled by the growth factor Flt3 ligand (Flt3L) ...and its receptor Flt3. We genetically dissected Flt3L-driven DC differentiation using CRISPR-Cas9-based screening. Genome-wide screening identified multiple regulators of DC differentiation including subunits of TSC and GATOR1 complexes, which restricted progenitor growth but enabled DC differentiation by inhibiting mTOR signaling. An orthogonal screen identified the transcriptional repressor Trim33 (TIF-1γ) as a regulator of DC differentiation. Conditional targeting in vivo revealed an essential role of Trim33 in the development of all DCs, but not of monocytes or granulocytes. In particular, deletion of Trim33 caused rapid loss of DC progenitors, pDCs, and the cross-presenting cDC1 subset. Trim33-deficient Flt3
progenitors up-regulated pro-inflammatory and macrophage-specific genes but failed to induce the DC differentiation program. Collectively, these data elucidate mechanisms that control Flt3L-driven differentiation of the entire DC lineage and identify Trim33 as its essential regulator.
Vanadium (V) is a transition metal adhered to suspended particles. Previous studies demonstrated that V inhalation causes oxidative stress in the ependymal epithelium, the choroid plexus on brain ...lateral ventricles and in the retina. Inhaled-V reaches the eye´s retina through the systemic circulation; however, its effect on the retina has not been widely studied. The Müller glial cell provides support and structure to the retina, facilitates synapses and regulates the microenvironment and neuronal metabolism. Hence, it is of great interest to study the effect of V exposure on the expression and localization of specific biomarkers on this cell.
Male CD-1 mice were exposed to V inhalation 1 h/twice/week for 4 and 8-Wk. Expression changes in the retina of Glial fibrillary acidic protein, highly expressed in Müller glial cell when retina is damaged, and Glutamine synthetase, important in preventing excitotoxicity in the retina, were analysed by immunohistochemistry.
Glial fibrillary acidic protein expression increased at 4-Wk of V inhalation compared to the control and decreased at 8-Wk of exposure. A time-dependent gradual reduction in glutamine synthetase expression was observed.
Changes in glial fibrillary acidic protein expression induced by V suggest retinal damage, whereas glutamine synthetase gradual reduction might indicate that photoreceptors, which produce most of the glutamine synthetase substrate in the retina, are degenerating, probably as a consequence of the oxidative stress induced by V.
Oncologic patients with covid 19: A mexican endeavor Daniela Shveid-Gerson; Alejandro Noguez-Ramos; Diana A. Villegas-Osorno ...
Revista medica del hospital general de mexico s.s.a,
10/2021, Letnik:
84, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Introduction: The coronavirus disease 2019 (COVID-19) pandemic is a worldwide challenge. There are few reports regarding its behavior in cancer patients. Materials and methods: Retrospective study ...including cancer patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the ABC Medical Center in Mexico City. We include general and oncological variables. We analyzed clinical features and treatment of COVID-19 and its outcomes such as hospitalization and death. Results: We report 86 patients with cancer and SARS-CoV-2 infection. The vast majority of patients 80 (93.1%) had a solid tumor while the most frequent primary tumor was breast 40 (46.5%) and lung 8 (9.3%). The clinical stage of patients was I in 22.1%, II in 16.3%, III in 31.4%, and IV in 24.4%. Antibiotics were used in 37 patients (43%) and corticosteroids in 32 (37.2%). Discussion: During disease evolution, 11 (12.8%) patients were hospitalized and 6 (7.0%) died. Variables of significant association with hospitalization include gender (men, odds ratio OR 5.6), previous cardiac disease (OR 25.1), and hematologic malignancy (OR 8.1). Associations with higher mortality rates were gender (men, OR 15), clinical Stage III/IV cancer (OR 11.3), type 2 diabetes mellitus (OR 14.7), previous cardiac disease (OR 19.2), and targeted therapy (OR 9.0). Conclusions: We found lower hospitalization and mortality rates compared to what had been previously reported both in Mexico around the globe. Men and patients with previous cardiac disease had a significant higher risk of hospitalization and death. Hematologic malignancies (lymphoma) were associated with higher hospitalization. Clinical Stage III/IV, targeted therapy, and type 2 diabetes mellitus showed a statistically significant association with mortality risk.
Introduction: The Coronavirus disease (COVID-19) pandemic is a worldwide challenge. There are few useful tools to predict patient outcomes. Identification of biomarkers able to predict progression of ...the disease could improve the treatment of these patients. Objective: The objective of the study was to identify biomarkers of disease progression among patients with severe COVID-19 pneumonia. Materials and methods: A retrospective cohort study was conducted among severe COVID-19 pneumonia patients hospitalized in the American British Cowdray Medical Center in Mexico City. Disease progression was defined as use of vasoactive amines, need of non-invasive or invasive mechanical ventilation or death. Studied biomarkers included neutrophil/lymphocyte index, lymphocyte/platelet Ratio, C reactive protein, procalcitonin, D Dimer, lactic dehydrogenase (LDH), ferritin, 25–OH–Vitamin D, and interleukin 6. Results: We report 46 patients with severe COVID-19 pneumonia. Mean age was 51 years, the majority of whom 30 (65%) male. Median hospitalization was 9 days. 23 (50%) of patients presented disease progression. Ferritin and LDH were strongly associated with disease progression among our cohort. In addition, age was associated with worst prognosis with a relative risk 4.5 (1.2-16.9, p = 0.003). Conclusions: Age, ferritin, and LDH were associated with disease progression among patients with severe COVID-19 pneumonia.
Gene regulation is a complex process through which the cell can control the levels of gene products that allow the cell to establish an identity, as well as respond to environmental changes such as ...stress. This control is exerted by many different players at its different stages, from transcription to post-translational modifcations.CRISPR technology has revolutionized the feld of genetic screening, allowing researchers to investigate thousands of transcripts simultaneously by abolishing their expression. Furthermore, recent expansions to the system have allowed not only reversible repression of gene expression (CRISPRi), but also activation of expression leading to gain-of-function type of analysis (CRISPRa). In this thesis, I examine the advantages and challenges of using pooled CRISPR screens to study the role of several gene regulators.First I describe a screen targeting transcription factors in mouse embryonic stem cells(mESCs), using the CRISPR-ko system, to identify those involved in pluripotency maintenance. I show that a small library is capable of capturing the majority of biological signal found in genome wide screens at a fraction of the size. Furthermore I show that smaller libraries are more adept at identifying genes with small effect size.I was also able to showcase the additional biological information gained from performing time-series screens by developing a novel method called CRISPTimeR. This tool is based on mixed linear models and allows to use information from a time-series experiment to identify CRISPR hits while having the additional feature of performing temporal classifcation for said hits.Next, I use the same mESCs system to study long non-coding RNAs (lncRNAs) relevant to pluripotency using the CRISPRi system. In this screen, I highlight the challenges of targeting lncRNAs, such as poor annotation and low expression levels. To account for the frst issue I suggest the use of a hand refned annotation of their transcription start sites. Additionally the low expression levels could be addressed by using a small library with a more specifc phenotypic readout, such as reporter gene activity, since a small effect size is expected.Finally, I describe a saturation screen that allows to target large genomic regions around the PHOX2B locus to identify putative cis-regulatory elements. This screen identifed CRISPRa responsive elements involved in regulating the expression of genes within thePHOX2BTAD. These regions were then matched with the genes they control using single-cell RNA-seq data.Overall, in this thesis, I show that CRISPR pooled screens are a valuable tool for the study of gene regulation. I highlight the challenges of targeting different regulatory elements and suggest modifcation to the planning, execution and analysis of screens to account for these challenges. Furthermore I introduced a new tool for the analysis of pooled CRISPR screens, suitable for coding and non-coding screens, and capable of analyzing time-series data in addition to two-time point experiments.
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e16105
Background: Gastric cancer (GC) and gastroesophageal junction cancer (GEJ) are the second most prevalent and lethal digestive malignancies worldwide. According to GLOBOCAN 2020, ...the incidence in Mexican population is 4.5% and represents 7% of all deaths. In GC and GEJ, the frequency of HER2 overexpression varies widely in the literature. Actually the blocking anti-HER2 is the standard therapy in GC and GEJ with overexpression HER2 according to the ToGa Trial. There are few studies that describe this population in mexican patients. Methods: We conducted a retrospective, observational analysis. Medical records of patients with GC or GEJ cancer from January 2015 to January 2021 were reviewed. A total of 91 patients were retrieved from the pathology database. Clinicalpathologic features collected were sex, age, pTNM stage (AJCC 8th edition), histology type, HER-2 status, metastases sites and treatment. The HER-2 determination was performed in all cases with immunohistochemistry (IHQ). Results: We found 10 patients with GC and GEJ with HER-2 overexpression by IHC, five patients with FISH positive and 2 of them with amplification by next generation sequence (FoundationOne). The median age was 56.9 years (27-68). There were more male patients (n = 8) than female patients (n = 2), with male/female ratio of 4:1. 8 patients had performance status (PS) 0 or 1, and the rest PS 2. The primary site was gastric carcinoma in 3 patients, and in 7 the tumor was located in GEJ. The clinical stage at diagnosis in 7 patients was metastatic, and 3 locally advanced. The most common sites of metastasis were lung (n = 5), liver (n = 4), and pleural, bone and central nervous system (each one with one patient). Intestinal-type GCs were most prevalent with 8 patients, and the rest were diffuse and signet ring cells. All patients received anti-HER2 blockade, 6 in the first line therapy, and 4 patients in the second line or more. Regarding the backbone regimen of the trastuzumab based therapy, fluoropyrimidine/platinum were the mainstay in 9 patients, and 1 with platinum and taxane scheme. Median number of cycles of trastuzumab was 8 (range, 4-24). The tumor responses were as follows: complete response (CR) 10%, partial response (PR) 30%, stable disease 50% and progressive disease 1 patient. The median progression-free survival was 6.6 months (1.3-23.1 months), and a median of overall survival was 21 months (6.4-51.5 months). The most frequently reported adverse events were diarrhoea in 3 patients (all grade 2), and mucositis in 1 patient. Cardiac adverse events were not reported. Conclusions: In our cohort, the prevalence of HER2 overexpression by IHC was 11%. In previous reports, the most important ToGa trial, 22% of the patients with metastatic GC and GEJ were HER2 positive. The PFS and OS in ToGa trial were 13.8 and 17.1 months respectively. By comparison our cohort the PFS and OS in ToGa trial were 6.6 and 21 months.
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e12574
Background: Currently there are no primary cultures or cell lines derived from patients with breast cancer and obesity. It has been postulated that breast cancer in obese women ...behaves differently as it does in non-obese women, as is composed of distinct biological features, as was generated in a different metabolic environment, as well as pertains to a different prognosis and different response to chemotherapy, lower rates of overall survival and a greater probability of recurrence. By creating a primary breast cancer culture bank of breast cancer tumors from women with obesity (BMI > 30kg/m
2
), we will establish a cell line exclusive to obese women in Mexico, where targeted therapy may be tested and treatment may be individualized depending on the characteristics of the patient. Methods: This study recruited 32 women with breast cancer and a BMI > 30 kg/m
2
, matched by 6 controls with are non-obese women with breast cancer. Elegibility criteria was determined by women with breast cancer confirmed by pathology, who had not been subjected to prior treatment regarding the neoplasm. The breast cancer removing surgeries and the patients were selected from the ABC Medical Center in Mexico City and all procedures were approved by the research and ethics committee of the hospital in question. Results: Through extensive communication a cooperative protocol was established between the departments of surgery, oncology, pathology and nursing to coordinate efforts and be able to take a 2 – 5 mm sample of the breast tumor removed from the patient. To be able to distinguish cancer cells from non-cancer cells (epithelial cells, fibroblasts, adipocytes) the Hayflick limit was be utilized. Once a primary breast cancer culture was established, 12 million cells will be injected into the subscapular area of athymic, nu-nu mice to be able to monitor tumoral growth in vivo and conduct a subsequent cellular analysis, determining it still pertains to the same characteristics of the tumor from which it was obtained. Conclusions: A primary breast cancer culture repository from patients with a BMI > 30 kg/m2 was established. This is the first primary breast cancer culture for both Mexican and obese women with breast cancer, the first in vitro method of analysis of specific characteristics typical of the Mexican population. Translational research may now be conducted on these new tumoral cultures to create individualized therapy for women with the distinct, aforementioned characteristics.
e21522
Background: Immune-related adverse effects (irAE´s) of immune-checkpoint inhibitors (ICIs) have been linked with a better treatment response in melanoma patients, especially cutaneous ...toxicities. However, little is known regarding other irAE´s which is important as they can be used as clinical markers of an adequate therapeutical response. Methods: We conducted a retrospective study on patients who were diagnosed with melanoma and received treatment with ICI´s between January 2015 until December 2021, immune related adverse events and their relationship with overall survival in melanoma patients treated with ICIs was the main objective of this study. Results: 53 records of patients with advance melanoma treated with ICIs between january 2016 to december 2021, demographic characteristics were as follow: 64.2% were male, mean age at diagnoses was 60.3 years, 41.5% had smoking history and 15.1% were Jewish. At diagnosis 73.6% of patients had a good functional status (ECOG 0-1). The most common histological subtypes were epithelioid (34%), and nodular (22.6%). Lung metastases was the most common affected site (49.1%), followed by brain 43.4% and non-regional nodes 42.5%. BRAF mutations was determined in 81.1% of the biopsies and 36% of them being V600E mutation. ICI´s was the preferred first line treatment in 83% of cases, median number of administered cycles were 6 (range 1-54 cycles), 60.4% of patients received pembrolizumab, 37.7% nivolumab plus ipilimumab, 20.8% nivolumab monotherapy and 5.7% ipilimumab. Throughout the studied period IrAE´s were reported in 34% of patients with 66.7% of them being grade 1-2 and 33.3% grade 3-4. The most common IrAE’s: vitiligo 38.8%, hypothyroidism22% and 3.8% pneumonitis. Median PFS at 12 months and OS was significantly better in the group of patients with irAE´s: Patients who develop an irAE´s are 7 times more likely to be disease free at 12 months and 4.1 times more likely to have a longer OS regardless of severity and type of toxicity. The impact of developing irAEs is significantly important for PFS (HR: 11.9, CI 95%: 3.28-4.71) as median PFS was not yet reached in this group. Conclusions: Development of irAEs is associated with favorable outcomes to ICIs with patients being 7 times more likely to be 12-month disease free and 4.1 times more likely to have a longer OS. irAEs can be used as clinical markers of an adequate treatment response.Table: see text
e22517
Background: there are few reported series n women with breast cancer (BC) and COVID-19, a better prognosis has been observed, with a lower rate of hospitalization and mortality than other ...neoplasms. Methods: We conducted a restrospective, non-experimental, observational, single center, study with a sample of 69 patients with BC who had presented COVID-19, in the period between March 2020 to August 2021. Clinicopathological characteristics of patients with BC were compared between severe and non-severe covid 19 groups, as well as hospitalized and non-hospitalized patients. An analysis of possible risk factors associated with severe disease and hospitalization was performed. Results: 69 cases were reported, median age 52y, mean BMI 25.2, ECOG 0-1: 97%. Smoking history in 24%, diabetes and hypertension were the most frequent comorbidities. The most frequent histology was ductal carcinoma in 80.6%, 73.8% showed ER + and 69.3% PR +, HER2 was overexpressed in 9.2%. The early stages predominated, I 22 (31.3%), II 25 (37.3%), III in 12 (17.9%) and IV in 6 (9%). The most frequents symptoms of COVID-19 were fatigue 70.1%, fever 65.7%, cough 59.7%, headache 56.7%, hyposmia 47.8%, dysgeusia 38.8%. A total of 53 (76.8%) mild cases, 14 (20.3) severe cases and 2 (2.9%) critical cases were registered. The 89.9% (62 patients) were treated as an outpatient basis, while 7 (10.1%) required hospitalization. Active treatment (< 45 days) at the time of COVID-19 was hormonal therapy 36 (50.7%), chemotherapy 11 (16.4%), anti-HER2 in 3 (4.5%), immunotherapy in 1.5%, targeted treatment in 4 (6.0%), surgery in 7 (10.4%) and radiotherapy in 1 (1.5%) patient. When comparing the severe and non-severe groups, as well as hospitalized versus non-hospitalized, we observed no difference between the clinicopathological characteristics. Then, we serch for possible risk factors, in wich, surgery in a period of less than 3 months increases the risk of severity OR 1,297 (95% CI 1,112-1,514), the risk of hospitalization increased in the triple negative subgroup OR 1,143 (95% CI, 1,035- 1,262), surgery less than 3 months OR 1,116 (1,014-1,229) and chemotherapy less than 45 days OR 1,217 (95% CI, 1,024-1,447). Conclusions: In patients with BC, the prevalence of severe or critical COVID-19 was 23% and the hospitalizacion rate 10%. No patient died from this infection. The clinical and pathological characteristics of BC do not appear to increase the risk of severe COVID-19 or the rate of hospitalization. Surgery performed in a period of less than 3 months is marginally associated with an increased risk of severe disease. Chemotherapy, targeted therapy, and immunotherapy do not modify the risk of severe disease; however, higher Ki 67, triple negative subgroup, surgery and chemotherapy showed a slight increase in risk of hospitalization.
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