Promoter Deletion Leading to Allele Specific Expression in a Genetically Unsolved Case of Primary Ciliary Dyskinesia.

Variation in the non-coding genome represents an understudied mechanism of disease and it remains challenging to predict if single nucleotide variants, small insertions and deletions, or structural variants in non-coding genomic regions will be detrimental. Our approach using complementary RNA-seq and targeted long-read DNA sequencing can prioritize identification of non-coding variants that lead to disease via alteration of gene splicing or expression. We have identified a patient with primary ciliary dyskinesia with a pathogenic coding variant on one allele of the SPAG1 gene, while the second allele appears normal by whole exome sequencing despite an autosomal recessive inheritance pattern. RNA sequencing revealed reduced SPAG1 transcript levels and exclusive allele specific expression of the known pathogenic allele, suggesting the presence of a non-coding variant on the second allele that impacts transcription. Targeted long-read DNA sequencing identified a heterozygous 3 kilobase deletion of the 5' untranslated region of SPAG1, overlapping the promoter and first non-coding exon. This non-coding deletion was missed by whole exome sequencing and gene-specific deletion/duplication analysis, highlighting the importance of investigating the non-coding genome in patients with "missing" disease-causing variation. This paradigm demonstrates the utility of both RNA and long-read DNA sequencing in identifying pathogenic non-coding variants in patients with unexplained genetic disease.

Characterization and bioinformatic filtering of ambient gRNAs in single-cell CRISPR screens using CLEANSER.

Recent technological developments in single-cell RNA-seq CRISPR screens enable high-throughput investigation of the genome. Through transduction of a gRNA library to a cell population followed by transcriptomic profiling by scRNA-seq, it is possible to characterize the effects of thousands of genomic perturbations on global gene expression. A major source of noise in scRNA-seq CRISPR screens are ambient gRNAs, which are contaminating gRNAs that likely originate from other cells. If not properly filtered, ambient gRNAs can result in an excess of false positive gRNA assignments. Here, we utilize CRISPR barnyard assays to characterize ambient gRNA noise in single-cell CRISPR screens. We use these datasets to develop and train CLEANSER, a mixture model that identifies and filters ambient gRNA noise. This model takes advantage of the bimodal distribution between native and ambient gRNAs and includes both gRNA and cell-specific normalization parameters, correcting for confounding technical factors that affect individual gRNAs and cells. The output of CLEANSER is the probability that a gRNA-cell assignment is in the native distribution over the ambient distribution. We find that ambient gRNA filtering methods impact differential gene expression analysis outcomes and that CLEANSER outperforms alternate approaches by increasing gRNA-cell assignment accuracy.

Synthesis, Characterization, and Biological Evaluation of Chitosan Nanoparticles Cross-Linked with Phytic Acid and Loaded with Colistin against Extensively Drug-Resistant Bacteria.

The natural evolution of microorganisms, as well as the inappropriate use of medicines, have accelerated the problem of drug resistance to many of the antibiotics employed today. Colistin, a lipopeptide antibiotic used as a last resort against multi-resistant strains, has also begun to present these challenges. Therefore, this study was focused on establishing whether colistin associated with chitosan nanoparticles could improve its antibiotic activity on an extremely resistant clinical isolate of Pseudomonas aeruginosa, which is a clinically relevant Gram-negative bacterium. For this aim, nanoparticulate systems based on phytic acid cross-linked chitosan and loaded with colistin were prepared by the ionic gelation method. The characterization included particle size, polydispersity index-PDI, and zeta potential measurements, as well as thermal (DSC) and spectrophotometric (FTIR) analysis. Encapsulation efficiency was assessed by the bicinchoninic acid (BCA) method, while the antimicrobial evaluation was made following the CLSI guidelines. The results showed that colistin-loaded nanoparticles were monodispersed (PDI = 0.196) with a particle size of around 266 nm and a positive zeta potential (+33.5 mV), and were able to associate with around 65.8% of colistin and decrease the minimum inhibitory concentration from 16 µg/mL to 4 µg/mL. These results suggest that the association of antibiotics with nanostructured systems could be an interesting alternative to recover the antimicrobial activity on resistant strains.

Descriptive study of the oral health status of disadvantaged mexican populations in relation to their adherence to the mediterranean diet.

BACKGROUND: Obesity in Mexico is an alarming problem that has been increasing in recent decades. Dietary factors make this pathology more common at younger ages and closely related to oral health. This study attempts to investigate the association between the oral health status of a Mexican population in the state of Yucatan and their dietary habits. OBJECTIVE: This study explores the relationship between oral health-related quality of life and adherence to the Mediterranean diet in a disadvantaged population in the state of Yucatan, Mexico. METHODS: The research was conducted in July 2023 in Merida, Yucatan (Mexico). The sample consisted of 109 individuals aged between 4 and 72 years old. Data analysis focused on factors such as body mass index (BMI), oral health-related quality of life, and adherence to the Mediterranean diet. RESULTS: A notable presence of caries is observed in individuals with low adherence to the Mediterranean diet (Correlation coefficient 0.040, p=0.682). This underscores the potential interaction between oral health, obesity, and dietary habits. The mean Oral Health-Related Quality of Life (OHIP-14Sp) score was 13.19 ±13.57, median 8.00. CONCLUSIONS: This research adds to the increasing evidence that highlights the significance of a balanced diet in enhancing the oral quality of life for people. More research is necessary to explore preventive measures and treatment to raise awareness about oral health within the community.

ANTECEDENTES: La obesidad en México es un problema alarmante que ha ido en aumento en las últimas décadas. Los factores dietéticos hacen que esta patología sea más común en edades más tempranas y muy relacionada con la salud bucal. Este estudio intenta investigar la asociación entre el estado de salud bucal de una población mexicana en el estado de Yucatán y sus hábitos alimentarios. OBJETIVO: Este estudio explora la relación entre la calidad de vida relacionada con la salud bucal y la adherencia a la dieta mediterránea en una población desfavorecida del estado de Yucatán, México. Métodos: La investigación se realizó en julio de 2023 en Mérida, Yucatán (México). La muestra estuvo compuesta por 109 individuos con edades comprendidas entre 4 y 72 años. El análisis de los datos se centró en factores como el índice de masa corporal (IMC), la calidad de vida relacionada con la salud bucal y la adherencia a la dieta mediterránea. RESULTADOS: Se observa una notable presencia de caries en individuos con baja adherencia a la dieta mediterránea (coeficiente de correlación 0,040, p=0,682). Esto subraya la posible interacción entre la salud bucal, la obesidad y los hábitos alimentarios. La puntuación media de la calidad de vida relacionada con la salud bucal (OHIP-14Sp) fue 13,19 ± 13,57, mediana 8,00. CONCLUSIONES: Esta investigación se suma a la creciente evidencia que resalta la importancia de una dieta equilibrada para mejorar la calidad de vida bucal de las personas. Es necesaria más investigación para explorar medidas preventivas y tratamientos para crear conciencia sobre la salud bucal dentro de la comunidad.

Mediator kinase inhibition impedes transcriptional plasticity and prevents resistance to ERK/MAPK-targeted therapy in KRAS-mutant cancers.

Acquired resistance remains a major challenge for therapies targeting oncogene activated pathways. KRAS is the most frequently mutated oncogene in human cancers, yet strategies targeting its downstream signaling kinases have failed to produce durable treatment responses. Here, we developed multiple models of acquired resistance to dual-mechanism ERK/MAPK inhibitors across KRAS-mutant pancreatic, colorectal, and lung cancers, and then probed the long-term events enabling survival against this class of drugs. These studies revealed that resistance emerges secondary to large-scale transcriptional adaptations that are diverse and cell line-specific. Transcriptional reprogramming extends beyond the well-established early response, and instead represents a dynamic, evolved process that is refined to attain a stably resistant phenotype. Mechanistic and translational studies reveal that resistance to dual-mechanism ERK/MAPK inhibition is broadly susceptible to manipulation of the epigenetic machinery, and that Mediator kinase, in particular, can be co-targeted at a bottleneck point to prevent diverse, cell line-specific resistance programs.

Machine learning methods for predicting guide RNA effects in CRISPR epigenome editing experiments.

CRISPR epigenomic editing technologies enable functional interrogation of non-coding elements. However, current computational methods for guide RNA (gRNA) design do not effectively predict the power potential, molecular and cellular impact to optimize for efficient gRNAs, which are crucial for successful applications of these technologies. We present "launch-dCas9" (machine LeArning based UNified CompreHensive framework for CRISPR-dCas9) to predict gRNA impact from multiple perspectives, including cell fitness, wildtype abundance (gauging power potential), and gene expression in single cells. Our launchdCas9, built and evaluated using experiments involving >1 million gRNAs targeted across the human genome, demonstrates relatively high prediction accuracy (AUC up to 0.81) and generalizes across cell lines. Method-prioritized top gRNA(s) are 4.6-fold more likely to exert effects, compared to other gRNAs in the same cis-regulatory region. Furthermore, launchdCas9 identifies the most critical sequence-related features and functional annotations from >40 features considered. Our results establish launch-dCas9 as a promising approach to design gRNAs for CRISPR epigenomic experiments.

Effectiveness of family metacognitive training in mothers with psychosis and their adolescent children: a multicenter study protocol.

Background: More than half of women with psychosis take care of their children despite the difficulties caused by the disease. Additionally, these kids have a higher risk of developing a mental health disorder. However, no interventions have been developed to meet these needs. Metacognitive Training (MCT) is a psychological intervention that has demonstrated its efficacy in improving cognitive insight, symptom management and social cognition in people with first-episode psychosis (FEP). Additionally, MCT has shown better results in women than men with FEP. This study aims to adapt and evaluate the efficacy of MCT-F in mothers and adolescent children in an online group context with the main purpose of improving family relationships, cognitive awareness and symptoms in women with psychosis and increase their children's knowledge of the disease and their functioning. As secondary objectives, it also aims to evaluate improvements in metacognition, social cognition, symptoms, protective factors and self-perception of stigma. Materials and methods: A quasi-experimental design with participants acting as their own control will be carried out. Forty-eight mothers with psychosis and their adolescent children (between 12 and 20 years old) recruited from a total of 11 adult mental health care centers will receive MCT-F. Participants will be evaluated 11 weeks before the intervention (T1), at baseline (T2), and post-intervention (T3) with a cognitive insight scale, as a primary outcome. Measures of metacognitive and social cognition, symptoms, cognitive functioning, family and social functioning, protective factors (self-esteem, resilience, and coping strategies) and self-perceived stigma will be addressed as secondary outcomes. Assessment will also address trauma and attachment in mothers and, lastly, the feasibility and acceptability of MCT-F in both participant groups. Discussion: This will be the first investigation of the efficacy, acceptability, and viability of the implementation of MCT-F. The results of this study may have clinical implications, contributing to improving mothers' with psychosis and adolescents' functioning and better understanding of the disease, in addition to the possible protective and preventive effect in adolescents, who are known to be at higher risk of developing severe mental disorders.Clinical trial registration:https://clinicaltrials.gov/, identifier [NCT05358457].

Multicenter integrated analysis of noncoding CRISPRi screens.

The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.

Activation of the imprinted Prader-Willi Syndrome locus by CRISPR-based epigenome editing.

Epigenome editing with DNA-targeting technologies such as CRISPR-dCas9 can be used to dissect gene regulatory mechanisms and potentially treat associated disorders. For example, Prader-Willi Syndrome (PWS) is caused by loss of paternally expressed imprinted genes on chromosome 15q11.2-q13.3, although the maternal allele is intact but epigenetically silenced. Using CRISPR repression and activation screens in human induced pluripotent stem cells (iPSCs), we identified genomic elements that control expression of the PWS gene SNRPN from the paternal and maternal chromosomes. We showed that either targeted transcriptional activation or DNA demethylation can activate the silenced maternal SNRPN and downstream PWS transcripts. However, these two approaches function at unique regions, preferentially activating different transcript variants and involving distinct epigenetic reprogramming mechanisms. Remarkably, transient expression of the targeted demethylase leads to stable, long-term maternal SNRPN expression in PWS iPSCs. This work uncovers targeted epigenetic manipulations to reprogram a disease-associated imprinted locus and suggests possible therapeutic interventions.

Mechanosensitive genomic enhancers potentiate the cellular response to matrix stiffness.

Epigenetic control of cellular transcription and phenotype is influenced by changes in the cellular microenvironment, yet how mechanical cues from these microenvironments precisely influence epigenetic state to regulate transcription remains largely unmapped. Here, we combine genome-wide epigenome profiling, epigenome editing, and phenotypic and single-cell RNA-seq CRISPR screening to identify a new class of genomic enhancers that responds to the mechanical microenvironment. These 'mechanoenhancers' could be active on either soft or stiff extracellular matrix contexts, and regulated transcription to influence critical cell functions including apoptosis, mechanotransduction, proliferation, and migration. Epigenetic editing of mechanoenhancers on rigid materials tuned gene expression to levels observed on softer materials, thereby reprogramming the cellular response to the mechanical microenvironment. These editing approaches may enable the precise alteration of mechanically-driven disease states.

Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens.

Clinical response to adoptive T cell therapies is associated with the transcriptional and epigenetic state of the cell product. Thus, discovery of regulators of T cell gene networks and their corresponding phenotypes has potential to improve T cell therapies. Here we developed pooled, epigenetic CRISPR screening approaches to systematically profile the effects of activating or repressing 120 transcriptional and epigenetic regulators on human CD8+ T cell state. We found that BATF3 overexpression promoted specific features of memory T cells and attenuated gene programs associated with cytotoxicity, regulatory T cell function, and exhaustion. Upon chronic antigen stimulation, BATF3 overexpression countered phenotypic and epigenetic signatures of T cell exhaustion. Moreover, BATF3 enhanced the potency of CAR T cells in both in vitro and in vivo tumor models and programmed a transcriptional profile that correlates with positive clinical response to adoptive T cell therapy. Finally, we performed CRISPR knockout screens that defined cofactors and downstream mediators of the BATF3 gene network.

Periodontal health of a low socioeconomic level population in Yucatan (Mexico): A cross-sectional study.

With the objective of assessing the periodontal health status, treatment needs, and oral hygiene habits of the population of Mérida, in Mexico, a descriptive cross-sectional study was performed. Four hundred forty individuals individually completed a questionnaire on oral health, oral hygiene habits, and quality of life. Additionally, a complete clinical dental examination was performed for each. For the statistical analysis, continuous variables (means and standard deviation) and categorical variables (frequencies) were studied. The associations were made using the analysis of variance test for continuous variables and the Chi-square test for categorical variables. The critical value to identify statistically significant differences was P < .05. The main concern of the population was the possible untreated caries they thought they had, with 36.21% followed by pain with 14.62%. Possible periodontal issues were the main discomfort for only 9%. The percentage of the sample that required periodontal intervention by a specialist was 21.14%. Statistically significant differences were found between age, place of residence, socioeconomic level, and schooling. There are great deficiencies in oral health in the studied group, which is accompanied by a great need for periodontal treatment. Periodontal health is closely related to oral hygiene, so the related sociocultural level should also be taken into account for the study of oral health in the most vulnerable populations. It is crucial to establish strategies to promote oral health.

Mediator Kinase Inhibition Impedes Transcriptional Plasticity and Prevents Resistance to ERK/MAPK-Targeted Therapy in KRAS-Mutant Cancers.

Acquired resistance remains a major challenge for therapies targeting oncogene activated pathways. KRAS is the most frequently mutated oncogene in human cancers, yet strategies targeting its downstream signaling kinases have failed to produce durable treatment responses. Here, we developed multiple models of acquired resistance to dual-mechanism ERK/MAPK inhibitors across KRAS-mutant pancreatic, colorectal, and lung cancers, and then probed the long-term events enabling survival against this class of drugs. These studies revealed that resistance emerges secondary to large-scale transcriptional adaptations that are diverse and cell line-specific. Transcriptional reprogramming extends beyond the well-established early response, and instead represents a dynamic, evolved process that is refined to attain a stably resistant phenotype. Mechanistic and translational studies reveal that resistance to dual-mechanism ERK/MAPK inhibition is broadly susceptible to manipulation of the epigenetic machinery, and that Mediator kinase, in particular, can be co-targeted at a bottleneck point to prevent diverse, cell line-specific resistance programs.

Integrin α3 promotes TH17 cell polarization and extravasation during autoimmune neuroinflammation.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by CNS-infiltrating leukocytes, including TH17 cells that are critical mediators of disease pathogenesis. Although targeting leukocyte trafficking is effective in treating autoimmunity, there are currently no therapeutic interventions that specifically block encephalitogenic TH17 cell migration. Here, we report integrin α3 as a TH17 cell-selective determinant of pathogenicity in experimental autoimmune encephalomyelitis. CNS-infiltrating TH17 cells express high integrin α3, and its deletion in CD4+ T cells or Il17a fate-mapped cells attenuated disease severity. Mechanistically, integrin α3 enhanced the immunological synapse formation to promote the polarization and proliferation of TH17 cells. Moreover, the transmigration of TH17 cells into the CNS was dependent on integrin α3, and integrin α3 deficiency enhanced the retention of CD4+ T cells in the perivascular space of the blood-brain barrier. Integrin α3-dependent interactions continuously maintain TH17 cell identity and effector function. The requirement of integrin α3 in TH17 cell pathogenicity suggests integrin α3 as a therapeutic target for MS treatment.

Cooperative regulation of coupled oncoprotein synthesis and stability in triple-negative breast cancer by EGFR and CDK12/13.

Evidence has long suggested that epidermal growth factor receptor (EGFR) may play a prominent role in triple-negative breast cancer (TNBC) pathogenesis, but clinical trials of EGFR inhibitors have yielded disappointing results. Using a candidate drug screen, we identified that inhibition of cyclin-dependent kinases 12 and 13 (CDK12/13) dramatically sensitizes diverse models of TNBC to EGFR blockade. This combination therapy drives cell death through the 4E-BP1-dependent suppression of the translation and translation-linked turnover of driver oncoproteins, including MYC. A genome-wide CRISPR/Cas9 screen identified the CCR4-NOT complex as a major determinant of sensitivity to the combination therapy whose loss renders 4E-BP1 unresponsive to drug-induced dephosphorylation, thereby rescuing MYC translational suppression and promoting MYC stability. The central roles of CCR4-NOT and 4E-BP1 in response to the combination therapy were further underscored by the observation of CNOT1 loss and rescue of 4E-BP1 phosphorylation in TNBC cells that naturally evolved therapy resistance. Thus, pharmacological inhibition of CDK12/13 reveals a long-proposed EGFR dependence in TNBC that functions through the cooperative regulation of translation-coupled oncoprotein stability.

Risk Factors Associated with COVID-19 Lethality: A Machine Learning Approach Using Mexico Database.

Identifying risk factors associated with COVID-19 lethality is crucial in combating the ongoing pandemic. In this study, we developed lethality predictive models for each epidemiological wave and for the overall dataset using the Extreme Gradient Boosting technique and analyzed them using Shapley values to determine the contribution levels of various features, including demographics, comorbidities, medical units, and recent medical information from confirmed COVID-19 cases in Mexico between February 23, 2020, and April 15, 2022. The results showed that pneumonia and advanced age were the most important factors predicting patient death in all cohorts. Additionally, the medical unit where the patient received care acted as a risk or protective factor. IMSS medical units were identified as high-risk factors in all cohorts, except in wave four, while SSA medical units generally were moderate protective factors. We also found that intubation was a high-risk factor in the first epidemiological wave and a moderate-risk factor in the following waves. Female gender was a protective factor of moderate-high importance in all cohorts, while being between 18 and 29 years old was a moderate protective factor and being between 50 and 59 years old was a moderate risk factor. Additionally, diabetes (all cohorts), obesity (third wave), and hypertension (fourth wave) were identified as moderate risk factors. Finally, residing in municipalities with the lowest Human Development Index level represented a moderate risk factor. In conclusion, this study identified several significant risk factors associated with COVID-19 lethality in Mexico, which could aid policymakers in developing targeted interventions to reduce mortality rates.

Orthogonal CRISPR screens to identify transcriptional and epigenetic regulators of human CD8 T cell function.

The clinical response to adoptive T cell therapies is strongly associated with transcriptional and epigenetic state. Thus, technologies to discover regulators of T cell gene networks and their corresponding phenotypes have great potential to improve the efficacy of T cell therapies. We developed pooled CRISPR screening approaches with compact epigenome editors to systematically profile the effects of activation and repression of 120 transcription factors and epigenetic modifiers on human CD8+ T cell state. These screens nominated known and novel regulators of T cell phenotypes with BATF3 emerging as a high confidence gene in both screens. We found that BATF3 overexpression promoted specific features of memory T cells such as increased IL7R expression and glycolytic capacity, while attenuating gene programs associated with cytotoxicity, regulatory T cell function, and T cell exhaustion. In the context of chronic antigen stimulation, BATF3 overexpression countered phenotypic and epigenetic signatures of T cell exhaustion. CAR T cells overexpressing BATF3 significantly outperformed control CAR T cells in both in vitro and in vivo tumor models. Moreover, we found that BATF3 programmed a transcriptional profile that correlated with positive clinical response to adoptive T cell therapy. Finally, we performed CRISPR knockout screens with and without BATF3 overexpression to define co-factors and downstream factors of BATF3, as well as other therapeutic targets. These screens pointed to a model where BATF3 interacts with JUNB and IRF4 to regulate gene expression and illuminated several other novel targets for further investigation.

Analysis and Evaluation of Dental Caries in a Mexican Population: A Descriptive Transversal Study.

Oral diseases are an important public health problem owing to their high prevalence and strong impact on people, particularly in disadvantaged populations. There is a strong relationship between the socioeconomic situation and the prevalence and severity of these diseases. Mexico is among the countries with a higher frequency range in oral diseases, highlighting dental caries, which affect more than 90% of the Mexican population. MATERIALS AND METHOD: A cross-sectional, descriptive, and observational study was carried out in 552 individuals who underwent a complete cariogenic clinical examination in different populations of the state of Yucatan. All individuals were evaluated after providing informed consent and with the consent of their legal guardians for those under legal age. We used the caries measurement methods described by the World Health Organization (WHO). Prevalence of caries, DMFT, and dft indexes were measured. Other aspects were also studied, such as oral habits and the use of public or private dental services. RESULTS: The prevalence of caries in permanent dentition was 84%. Moreover, it was found to be statistically related to the following variables: place of residence, socioeconomic level, gender, and level of education (p < 0.05). For primary teeth, the prevalence was 64% and there was no statistical relation with any of the variables studied (p > 0.05). Regarding the other aspects studied, more than 50% of the sample used private dental services. CONCLUSIONS: There is a high need for dental treatment in the population studied. It is necessary to develop prevention and treatment strategies considering the particularities of each population, driving collaborative projects to promote better oral health conditions in disadvantaged populations.