LncRNA DCRT Protects Against Dilated Cardiomyopathy by Preventing NDUFS2 Alternative Splicing by Binding to PTBP1.

BACKGROUND: Dilated cardiomyopathy is characterized by left ventricular dilation and continuous systolic dysfunction. Mitochondrial impairment is critical in dilated cardiomyopathy; however, the underlying mechanisms remain unclear. Here, we explored the cardioprotective role of a heart-enriched long noncoding RNA, the dilated cardiomyopathy repressive transcript (DCRT), in maintaining mitochondrial function. METHODS: The DCRT knockout (DCRT-/-) mice and DCRT knockout cells were developed using CRISPR-Cas9 technology. Cardiac-specific DCRT transgenic mice were generated using α-myosin heavy chain promoter. Chromatin coimmunoprecipitation, RNA immunoprecipitation, Western blot, and isoform sequencing were performed to investigate the underlying mechanisms. RESULTS: We found that the long noncoding RNA DCRT was highly enriched in the normal heart tissues and that its expression was significantly downregulated in the myocardium of patients with dilated cardiomyopathy. DCRT-/- mice spontaneously developed cardiac dysfunction and enlargement with mitochondrial impairment. DCRT transgene or overexpression with the recombinant adeno-associated virus system in mice attenuated cardiac dysfunction induced by transverse aortic constriction treatment. Mechanistically, DCRT inhibited the third exon skipping of NDUFS2 (NADH dehydrogenase ubiquinone iron-sulfur protein 2) by directly binding to PTBP1 (polypyrimidine tract binding protein 1) in the nucleus of cardiomyocytes. Skipping of the third exon of NDUFS2 induced mitochondrial dysfunction by competitively inhibiting mitochondrial complex I activity and binding to PRDX5 (peroxiredoxin 5) and suppressing its antioxidant activity. Furthermore, coenzyme Q10 partially alleviated mitochondrial dysfunction in cardiomyocytes caused by DCRT reduction. CONCLUSIONS: Our study revealed that the loss of DCRT contributed to PTBP1-mediated exon skipping of NDUFS2, thereby inducing cardiac mitochondrial dysfunction during dilated cardiomyopathy development, which could be partially treated with coenzyme Q10 supplementation.

Genome-scale exon perturbation screens uncover exons critical for cell fitness.

CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit TAF5 alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression.

The unique properties of Big tau in the visual system.

Tau is a microtubule associated protein that plays important roles in regulating the properties of microtubules and axonal transport, as well as tauopathies associated with toxic aggregates leading to neurodegenerative diseases. It is encoded by the MAPT gene forming multiple isoforms (45-60 kDa) by alternative splicing which are developmentally regulated. The high molecular weight (MW) tau isoform of 105 kDa, termed Big tau, was originally discovered in the peripheral nervous system (PNS) but later found in selective CNS areas. It contains an additional large exon 4a generating a long projecting domain of about 250 amino acids. Here we investigated the properties of Big tau in the visual system of rats, its distribution in retinal ganglion cells and the optic nerve as well as its developmental regulation using biochemical, molecular and histological analyses. We discovered that Big tau is expresses as a 95 kDa protein (termed middle MW) containing exons 4a, 6 as well as exon 10 which defines a 4 microtubule-binding repeats (4R). It lacks exons 2/3 but shares the extensive phosphorylation characteristic of other tau isoforms. Importantly, early in development the visual system expresses only the low MW isoform (3R) switching to both the low and middle MW isoforms (4R) in adult retinal ganglion neurons and their corresponding axons. This is a unique structure and expression pattern of Big tau, which we hypothesize is associated with the specific properties of the visual system different from what has been previously described in the PNS and other areas of the nervous system.

Abundant transcriptomic alterations in the human cerebellum of patients with a C9orf72 repeat expansion.

The most prominent genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) is a repeat expansion in the gene C9orf72. Importantly, the transcriptomic consequences of the C9orf72 repeat expansion remain largely unclear. Here, we used short-read RNA sequencing (RNAseq) to profile the cerebellar transcriptome, detecting alterations in patients with a C9orf72 repeat expansion. We focused on the cerebellum, since key C9orf72-related pathologies are abundant in this neuroanatomical region, yet TDP-43 pathology and neuronal loss are minimal. Consistent with previous work, we showed a reduction in the expression of the C9orf72 gene and an elevation in homeobox genes, when comparing patients with the expansion to both patients without the C9orf72 repeat expansion and control subjects. Interestingly, we identified more than 1000 alternative splicing events, including 4 in genes previously associated with ALS and/or FTLD. We also found an increase of cryptic splicing in C9orf72 patients compared to patients without the expansion and controls. Furthermore, we demonstrated that the expression level of select RNA-binding proteins is associated with cryptic splice junction inclusion. Overall, this study explores the presence of widespread transcriptomic changes in the cerebellum, a region not confounded by severe neurodegeneration, in post-mortem tissue from C9orf72 patients.

Differential expression of PPP1R12A transcripts, including those harbouring alternatively spliced micro-exons, in placentae from complicated pregnancies.

INTRODUCTION: Placenta-associated pregnancy complications, including pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are conditions postulated to originate from initial failure of placentation, leading to clinical sequelae indicative of endothelial dysfunction. Vascular smooth muscle aberrations have also been implicated in the pathogenesis of both disorders via smooth muscle contractility and relaxation mediated by Myosin Light Chain Phosphatase (MLCP) and the oppositional contractile action of Myosin Light Chain Kinase. PPP1R12A is a constituent part of the MLCP complex responsible for dephosphorylation of myosin fibrils. We hypothesize that alternative splicing of micro-exons result in isoforms lacking the functional leucine zipper (LZ) domain which may give those cells expressing these alternative transcripts a tendency towards contraction and vasoconstriction. METHODS: Expression was determined by qRT-PCR. Epigenetic profiling consisted of bisulphite-based DNA methylation analysis and ChIP for underlying histone modifications. RESULTS: We identified several novel transcripts with alternative micro-exon inclusion that would produce LZ- PPP1R12A protein. qRT-PCR revealed some isoforms, including the PPP1R12A canonical transcript, are differentially expressed in placenta biopsies from PE and IUGR samples compared to uncomplicated pregnancies. DISCUSSION: We propose that upregulation of PPP1R12A expression in complicated pregnancies may be due to enhanced promoter activity leading to increased transcription as a response to physiological stress in the placenta, which we show is independent of promoter DNA methylation.

Microsatellite Content in 397 Nuclear Exons and Their Flanking Regions in the Fern Family Ophioglossaceae.

Microsatellites or SSRs are small tandem repeats that are 1-6 bp long. They are usually highly polymorphic and form important portions of genomes. They have been extensively analyzed in humans, animals and model plants; however, information from non-flowering plants is generally lacking. Here, we examined 29 samples of Ophioglossaceae ferns, mainly from the genera Botrychium and Sceptridium. We analyzed the SSR distribution, density and composition in almost 400 nuclear exons and their flanking regions. We detected 45 SSRs in exons and 1475 SSRs in the flanking regions. In the exons, only di-, tri- and tetranucleotides were found, and all of them were 12 bp long. The annotation of the exons containing SSRs showed that they were related to various processes, such as metabolism, catalysis, transportation or plant growth. The flanking regions contained SSRs from all categories, with the most numerous being dinucleotides, followed by tetranucleotides. More than one-third of all the SSRs in the flanking regions were 12 bp long. The SSR densities in the exons were very low, ranging from 0 to 0.07 SSRs/kb, while those in the flanking regions ranged from 0.24 to 0.81 SSRs/kb; and those in the combined dataset ranged from 0.2 to 0.81 SSRs/kb. The majority of the detected SSRs in the flanking regions were polymorphic and present at the same loci across two or more samples but differing in the number of repeats. The SSRs detected here may serve as a basis for further population genetic, phylogenetic or evolutionary genetic studies, as well as for further studies focusing on SSRs in the genomes and their roles in adaptation, evolution and diseases.

Human exons and introns classification using pre-trained Resnet-50 and GoogleNet models and 13-layers CNN model.

BACKGROUND: Examining functions and characteristics of DNA sequences is a highly challenging task. When it comes to the human genome, which is made up of exons and introns, this task is more challenging. Human exons and introns contain millions to billions of nucleotides, which contributes to the complexity observed in this sequences. Considering how complicated the subject of genomics is, it is obvious that using signal processing techniques and deep learning tools to build a strong predictive model can be very helpful for the development of the research of the human genome. RESULTS: After representing human exons and introns with color images using Frequency Chaos Game Representation, two pre-trained convolutional neural network models (Resnet-50 and GoogleNet) and a proposed CNN model having 13 hidden layers were used to classify our obtained images. We have reached a value of 92% for the accuracy rate for Resnet-50 model in about 7 h for the execution time, a value of 91.5% for the accuracy rate for the GoogleNet model in 2 h and a half for the execution time. For our proposed CNN model, we have reached 91.6% for the accuracy rate in 2 h and 37 min. CONCLUSIONS: Our proposed CNN model is faster than the Resnet-50 model in terms of execution time. It was able to slightly exceed the GoogleNet model for the accuracy rate value.

Optimization strategies and advances in the research and development of AAV-based gene therapy to deliver large transgenes.

Adeno-associated virus (AAV)-based therapies are recognized as one of the most potent next-generation treatments for inherited and genetic diseases. However, several biological and technological aspects of AAV vectors remain a critical issue for their widespread clinical application. Among them, the limited capacity of the AAV genome significantly hinders the development of AAV-based gene therapy. In this context, genetically modified transgenes compatible with AAV are opening up new opportunities for unlimited gene therapies for many genetic disorders. Recent advances in de novo protein design and remodelling are paving the way for new, more efficient and targeted gene therapeutics. Using computational and genetic tools, AAV expression cassette and transgenic DNA can be split, miniaturized, shuffled or created from scratch to mediate efficient gene transfer into targeted cells. In this review, we highlight recent advances in AAV-based gene therapy with a focus on its use in translational research. We summarize recent research and development in gene therapy, with an emphasis on large transgenes (>4.8 kb) and optimizing strategies applied by biomedical companies in the research pipeline. We critically discuss the prospects for AAV-based treatment and some emerging challenges. We anticipate that the continued development of novel computational tools will lead to rapid advances in basic gene therapy research and translational studies.

Molecular and immunohistochemical study of APC exon 16 and its possible role in colorectal carcinoma development.

Background: Colorectal cancer ranks second as a cause of cancer deaths. Mutations in the adenomatous polyposis coli (APC) gene, especially in exon 16, could contribute to colorectal carcinoma development. This study explored the correlations between APC gene exon 16 variations/expression and colorectal carcinoma progression. Methods: In a case-control study, blood samples from 150 colorectal carcinoma patients and 50 healthy volunteers were analyzed by PCR and sequencing for APC exon 16 variations. The APC protein expression on tissue samples was evaluated by immunohistochemistry and statistical analyses were used to examine clinicopathological correlations. Results: The sequencing analysis revealed a mutation in exon 16 of the APC gene (rs459552) in 36 % of colorectal cancer cases while absent in all non-cancer controls. Subgroup analysis by tumor grade showed higher prevalence of mutant allele in Grade II and Grade III cases, with frequencies reaching 60.0 % and 69.2 %, respectively, compared to a substantially lower prevalence of 29.4 % in Grade I patients. Immunohistochemistry showed no significant correlation between this mutation and APC expression. APC positivity proportions were 25.5 % in Grade I tumors (n = 26/102) versus 17.1 % in Grade II (n = 6/35) and 46.2 % in Grade III (n = 6/13), showing a non-significant trend of reduced positivity in higher grade tumors (p>0.05). Conclusions: The frequency of APC exon 16 mutation (rs459552) rose significantly with increasing tumor grade. Similarly, although not statistically significant, the percentage of APC positive staining increased with poorer tumor differentiation, rather than declining. Therefore, the APC exon 16 mutation and expression analysis provides insights into colorectal cancer progression, with the rs459552 mutation correlating with grade and may promoting aggression.

Cryptic exon inclusion is a molecular signature of LATE-NC in aging brains.

The aggregation, mislocalization, and phosphorylation of TDP-43 are pathologic hallmarks of several neurodegenerative diseases and provide a defining criterion for the neuropathologic diagnosis of Limbic-predominant Age-related TDP-43 Encephalopathy (LATE). LATE neuropathologic changes (LATE-NC) are often comorbid with other neurodegenerative pathologies including Alzheimer's disease neuropathologic changes (ADNC). We examined whether TDP-43 regulated cryptic exons accumulate in the hippocampus of neuropathologically confirmed LATE-NC cases. We found that several cryptic RNAs are robustly expressed in LATE-NC cases with or without comorbid ADNC and correlate with pTDP-43 abundance; however, the accumulation of cryptic RNAs is more robust in LATE-NC with comorbid ADNC. Additionally, cryptic RNAs can robustly distinguish LATE-NC from healthy controls and AD cases. These findings expand our current understanding and provide novel potential biomarkers for LATE pathogenesis.

Cryptic splicing of stathmin-2 and UNC13A mRNAs is a pathological hallmark of TDP-43-associated Alzheimer's disease.

Nuclear clearance and cytoplasmic accumulations of the RNA-binding protein TDP-43 are pathological hallmarks in almost all patients with amyotrophic lateral sclerosis (ALS) and up to 50% of patients with frontotemporal dementia (FTD) and Alzheimer's disease. In Alzheimer's disease, TDP-43 pathology is predominantly observed in the limbic system and correlates with cognitive decline and reduced hippocampal volume. Disruption of nuclear TDP-43 function leads to abnormal RNA splicing and incorporation of erroneous cryptic exons in numerous transcripts including Stathmin-2 (STMN2, also known as SCG10) and UNC13A, recently reported in tissues from patients with ALS and FTD. Here, we identify both STMN2 and UNC13A cryptic exons in Alzheimer's disease patients, that correlate with TDP-43 pathology burden, but not with amyloid-ß or tau deposits. We also demonstrate that processing of the STMN2 pre-mRNA is more sensitive to TDP-43 loss of function than UNC13A. In addition, full-length RNAs encoding STMN2 and UNC13A are suppressed in large RNA-seq datasets generated from Alzheimer's disease post-mortem brain tissue. Collectively, these results open exciting new avenues to use STMN2 and UNC13A as potential therapeutic targets in a broad range of neurodegenerative conditions with TDP-43 proteinopathy including Alzheimer's disease.

Matching-adjusted indirect comparison of amivantamab vs mobocertinib in platinum-pretreated EGFR Exon 20 insertion-mutated non-small-cell lung cancer.

Aim: We assessed relative efficacy and safety of amivantamab versus mobocertinib in patients with non-small-cell lung cancer with EGFR exon 20 insertion (exon20ins) mutations who progressed on prior platinum-based chemotherapy. Materials & methods: This matching-adjusted indirect comparison used patient-level data from CHRYSALIS (NCT02609776) and aggregate data from a mobocertinib trial (NCT02716116) to match populations on all clinically relevant confounders. Results: While both agents had similar efficacy for time-to-event outcomes, objective response rate was significantly higher for amivantamab. 15 of 23 any-grade treatment-related adverse events reported for mobocertinib were significantly less common for amivantamab versus only two for mobocertinib. Conclusion: Results suggest that amivantamab has an improved response rate with similar survival and a more favorable safety profile versus mobocertinib in EGFR exon20ins non-small-cell lung cancer.

Transcriptional, biochemical, and immunohistochemical analyses of CaMKKß/2 splice variants that co-localize with CaMKIV in spermatids.

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates downstream protein kinases, including CaMKI, CaMKIV, PKB/Akt, and AMPK; thus, regulates various Ca2+-dependent physiological and pathophysiological pathways. Further, CaMKKß/2 in mammalian species comprises multiple alternatively spliced variants; however, their functional differences or redundancy remain unclear. In this study, we aimed to characterize mouse CaMKKß/2 splice variants (CaMKKß-3 and ß-3x). RT-PCR analyses revealed that mouse CaMKKß-1, consisting of 17 exons, was predominantly expressed in the brain; whereas, mouse CaMKKß-3 and ß-3x, lacking exon 16 and exons 14/16, respectively, were primarily expressed in peripheral tissues. At the protein level, the CaMKKß-3 or ß-3x variants showed high expression levels in mouse cerebrum and testes. This was consistent with the localization of CaMKKß-3/-3x in spermatids in seminiferous tubules, but not the localization of CaMKKß-1. We also observed the co-localization of CaMKKß-3/-3x with a target kinase, CaMKIV, in elongating spermatids. Biochemical characterization further revealed that CaMKKß-3 exhibited Ca2+/CaM-induced kinase activity similar to CaMKKß-1. Conversely, we noted that CaMKKß-3x impaired Ca2+/CaM-binding ability, but exhibited significantly weak autonomous activity (approximately 500-fold lower than CaMKKß-1 or ß-3) due to the absence of C-terminal of the catalytic domain and a putative residue (Ile478) responsible for the kinase autoinhibition. Nevertheless, CaMKKß-3x showed the ability to phosphorylate downstream kinases, including CaMKIα, CaMKIV, and AMPKα in transfected cells comparable to CaMKKß-1 and ß-3. Collectively, CaMKKß-3/-3x were identified as functionally active and could be bona fide CaMKIV-kinases in testes involved in the activation of the CaMKIV cascade in spermatids, resulting in the regulation of spermiogenesis.

The new missense G376V-TDP-43 variant induces late-onset distal myopathy but not amyotrophic lateral sclerosis.

TDP-43-positive inclusions in neurons are a hallmark of several neurodegenerative diseases including familial amyotrophic lateral sclerosis (fALS) caused by pathogenic TARDBP variants as well as more common non-Mendelian sporadic ALS (sALS). Here we report a G376V-TDP-43 missense variant in the C-terminal prion-like domain of the protein in two French families affected by an autosomal dominant myopathy but not fulfilling diagnostic criteria for ALS. Patients from both families presented with progressive weakness and atrophy of distal muscles, starting in their 5th-7th decade. Muscle biopsies revealed a degenerative myopathy characterized by accumulation of rimmed (autophagic) vacuoles, disruption of sarcomere integrity and severe myofibrillar disorganization. The G376 V variant altered a highly conserved amino acid residue and was absent in databases on human genome variation. Variant pathogenicity was supported by in silico analyses and functional studies. The G376 V mutant increased the formation of cytoplasmic TDP-43 condensates in cell culture models, promoted assembly into high molecular weight oligomers and aggregates in vitro, and altered morphology of TDP-43 condensates arising from phase separation. Moreover, the variant led to the formation of cytoplasmic TDP-43 condensates in patient-derived myoblasts and induced abnormal mRNA splicing in patient muscle tissue. The identification of individuals with TDP-43-related myopathy but not ALS implies that TARDBP missense variants may have more pleiotropic effects than previously anticipated and support a primary role for TDP-43 in skeletal muscle pathophysiology. We propose to include TARDBP screening in the genetic work-up of patients with late-onset distal myopathy. Further research is warranted to examine the precise pathogenic mechanisms of TARDBP variants causing either a neurodegenerative or myopathic phenotype.

Loss of TDP-43 splicing repression occurs early in the aging population and is associated with Alzheimer's disease neuropathologic changes and cognitive decline.

LATE-NC, the neuropathologic changes of limbic-predominant age-related TAR DNA-binding protein 43 kDa (TDP-43) encephalopathy are frequently associated with Alzheimer's disease (AD) and cognitive impairment in older adults. The association of TDP-43 proteinopathy with AD neuropathologic changes (ADNC) and its impact on specific cognitive domains are not fully understood and whether loss of TDP-43 function occurs early in the aging brain remains unknown. Here, using a large set of autopsies from the Baltimore Longitudinal Study of Aging (BLSA) and another younger cohort, we were able to study brains from subjects 21-109 years of age. Examination of these brains show that loss of TDP-43 splicing repression, as judged by TDP-43 nuclear clearance and expression of a cryptic exon in HDGFL2, first occurs during the 6th decade, preceding by a decade the appearance of TDP-43+ neuronal cytoplasmic inclusions (NCIs). We corroborated this observation using a monoclonal antibody to demonstrate a cryptic exon-encoded neoepitope within HDGFL2 in neurons exhibiting nuclear clearance of TDP-43. TDP-43 nuclear clearance is associated with increased burden of tau pathology. Age at death, female sex, high CERAD neuritic plaque score, and high Braak neurofibrillary stage significantly increase the odds of LATE-NC. Faster rates of cognitive decline on verbal memory (California Verbal Learning Test immediate recall), visuospatial ability (Card Rotations Test), mental status (MMSE) and semantic fluency (Category Fluency Test) were associated with LATE-NC. Notably, the effects of LATE-NC on verbal memory and visuospatial ability are independent of ADNC. However, the effects of TDP-43 nuclear clearance in absence of NCI on the longitudinal trajectories and levels of cognitive measures are not significant. These results establish that loss of TDP-43 splicing repression is an early event occurring in the aging population during the development of TDP-43 proteinopathy and is associated with increased tau pathology. Furthermore, LATE-NC correlates with high levels of ADNC but also has an impact on specific memory and visuospatial functions in aging that is independent of AD.

Genotype-phenotype spectrum and prognosis of early-onset Marfan syndrome.

BACKGROUND: Marfan syndrome is a genetic connective tissue disorder affecting skeletal, ocular, and cardiovascular organ systems. Previous research found that pathogenic variants clustered in exons 24-32 of fibrillin-1 (FBN1) gene result in more severe clinical phenotypes. Furthermore, genotype-phenotype correlation studies suggested that more severe cardiovascular phenotypes were related to variants held responsible for haploinsufficiency. Our objective was to analyze the differences in clinical manifestations and genotypes of individuals with early-onset Marfan syndrome and to assess their impact on management strategies. METHODS: We analyzed clinical and genetic data of a new patient with early-onset Marfan syndrome together with 51 previously reported ones in the PubMed database between 1991 and 2022. RESULTS: Analysis showed 94% (49/52) of pathogenic variants clustered in exons 24-32 of the FBN1. The most common skeletal features were arachnodactyly (98%), reduced elbow extension (48%), pectus deformity (40%), and scoliosis (39%). Haploinsufficiency variants were reported as having poor outcome in 87.5% of the cases. Among patients carrying variants that substitute a cysteine for another amino acid and those that do not change cysteine content, cardiac intervention was found to be associated with a better outcome (p = 0.035 vs. p = 0.002). Variants that create an extra cysteine residue were found to be associated with a higher risk of ectopia lentis. Additionally, children up to 36-months-old were more often reported as still alive at the time of publication compared to newborns (p < 0.01). CONCLUSIONS: Our findings have implications for prognosis, because different genotype groups and their resulting phenotype may require personalized care and management.

Mechanistic Insights into Alternative Gene Splicing in Oxidative Stress and Tissue Injury.

Significance: Oxidative stress (OS) and inflammation are inducers of tissue injury. Alternative splicing (AS) is an essential regulatory step for diversifying the eukaryotic proteome. Human diseases link AS to OS; however, the underlying mechanisms must be better understood. Recent Advances: Genome­wide profiling studies identify new differentially expressed genes induced by OS-dependent ischemia/reperfusion injury. Overexpression of RNA-binding protein RBFOX1 protects against inflammation. Hypoxia-inducible factor-1α directs polypyrimidine tract binding protein 1 to regulate mouse carcinoembryonic antigen-related cell adhesion molecule 1 (Ceacam1) AS under OS conditions. Heterogeneous nuclear ribonucleoprotein L variant 1 contains an RGG/RG motif that coordinates with transcription factors to influence human CEACAM1 AS. Hypoxia intervention involving short interfering RNAs directed to long-noncoding RNA 260 polarizes M2 macrophages toward an anti-inflammatory phenotype and alleviates OS by inhibiting IL-28RA gene AS. Critical Issues: Protective mechanisms that eliminate reactive oxygen species (ROS) are important for resolving imbalances that lead to chronic inflammation. Defects in AS can cause ROS generation, cell death regulation, and the activation of innate and adaptive immune factors. We propose that AS pathways link redox regulation to the activation or suppression of the inflammatory response during cellular stress. Future Directions: Emergent studies using molecule-mediated RNA splicing are being conducted to exploit the immunogenicity of AS protein products. Deciphering the mechanisms that connect misspliced OS and pathologies should remain a priority. Controlled release of RNA directly into cells with clinical applications is needed as the demand for innovative nucleic acid delivery systems continues to be demonstrated.

Reversing T Cell Exhaustion by Converting Membrane PD-1 to Its Soluble form in Jurkat Cells; Applying The CRISPR/Cas9 Exon Skipping Strategy.

OBJECTIVE: T-cells express two functional forms of the programmed cell death protein 1 (PD-1): membrane (mPD-1) and soluble (sPD-1). The binding of mPD-1 and its ligand (PD-L1) on tumor cells could lead activated lymphocytes toward exhaustion. Selective deletion of the transmembrane domain via alternative splicing of exon-3 in PD-1 mRNA could generate sPD-1. Overexpression of sPD-1 could disrupt the mPD-1/PD-L1 interaction in tumor-specific T cells. We investigated the effect of secreted sPD-1 from pooled engineered and non-engineered T cell supernatant on survival and proliferation of lymphocytes in the tumor microenvironment (TME). MATERIALS AND METHODS: In this experimental study, we designed two sgRNA sequences upstream and downstream of exon-3 in the PDCD1 gene. The lentiCRISPRv2 puro vector was used to clone the dual sgRNAs and produce lentiviral particles to transduce Jurkat T cells. Analysis assays were used to clarify the change in PD-1 expression pattern in the pooled (engineered and non-engineered) Jurkat cells. Co-culture conditions were established with PD-L1+ cancer cells and lymphocytes. RESULTS: CRISPR/Cas9 could delete exon-3 of the PDCD1 gene in the engineered cells based on the tracking of indels by decomposition (TIDE) and interference of CRISPR edit (ICE) sequencing analysis reports. Our results showed a 12% reduction in mPD-1 positive cell population after CRISPR manipulation and increment in sPD-1 concentration in the supernatant. The increased sPD-1 confirmed its positive effect on proliferation of lymphocytes co-cultured with PDL1+ cancer cells. The survival percent of lymphocytes co-cultured with the pooled cells supernatant was 12.5% more than the control. CONCLUSION: The CRISPR/Cas9 exon skipping approach could be used in adoptive cell immunotherapies to change PD-1 expression patterns and overcome exhaustion.

Familial Partial Lipodystrophy: Clinical Features, Genetics and Treatment in a Greek Referral Center.

Familial partial lipodystrophy (FPLD) is a rare syndrome in which a patient's phenotype is not merely dependent on the specific genetic mutation, but it is also defined by a combination of other demographic, environmental and genetic factors. In this prospective observational study in a Greek referral center, we enrolled 39 patients who fulfilled the clinical criteria of FPLD. A genetic analysis was conducted, which included sequence and deletion/duplication analyses of the LMNA and PPRARG genes, along with anthropometric and metabolic parameters. The treatment responses of patients who were eligible for treatment with metreleptin were evaluated at 3 and 12 months. In most of the patients, no significant changes were detected at the exon level, and any mutations that led to changes at the protein level were not associated with the lipodystrophic phenotype. On the contrary, various changes were detected at the intron level, especially in introns 7 and 10, whose clinical significance is considered unknown. In addition, treatment with metreleptin in specific FPLD patients significantly improved glycemic and lipidemic control, an effect which was sustained at the 12-month follow-up. More large-scale studies are necessary to clarify the genetic and allelic heterogeneity of the disease, along with other parameters which could predict treatment response.

Low hybridization temperatures improve target capture success of invertebrate loci: a case study of leaf-footed bugs (Hemiptera: Coreoidea).

Target capture is widely used in phylogenomic, ecological and functional genomic studies. Bait sets that allow capture from a diversity of species can be advantageous, but high-sequence divergence from baits can limit yields. Currently, only four experimental comparisons of a critical target capture parameter, hybridization temperature, have been published. These have been in vertebrates, where bait divergences are typically low, and none include invertebrates where bait-target divergences may be higher. Most invertebrate capture studies use a fixed, high hybridization temperature to maximize the proportion of on-target data, but many report low locus recovery. Using leaf-footed bugs (Hemiptera: Coreoidea), we investigate the effect of hybridization temperature on capture success of ultraconserved elements targeted by (i) baits developed from divergent hemipteran genomes and (ii) baits developed from less divergent coreoid transcriptomes. Lower temperatures generally resulted in more contigs and improved recovery of targets despite a lower proportion of on-target reads, lower read depth and more putative paralogues. Hybridization temperatures had less of an effect when using transcriptome-derived baits, which is probably due to lower bait-target divergences and greater bait tiling density. Thus, accommodating low hybridization temperatures during target capture can provide a cost-effective, widely applicable solution to improve invertebrate locus recovery.