DNA Nucleotide Research Articles

Immune checkpoint VISTA regulates DNA sensing and repair pathways that contribute to type I interferon (IFN-I) production in the skin

Abstract Skin sterile injury by ultraviolet (UV) light stimulates production of type I interferons. While in healthy skin IFN-I response to UV returns to baseline over time, the IFN-I signature persists in photosensitive diseases like lupus. The pathways that downregulate IFN-I production in healthy skin, and fail to do so in lupus skin, are unknown. Previous studies showing that activating VISTA suppresses the IFN-I signature in monocytes suggest a role for this immune checkpoint in the regulation of the IFN-I response. Indeed, we showed that the skin IFN-I signature is elevated in mice globally deficient in VISTA (Vsir−/−), as well as in mice with a conditional deletion of VISTA in keratinocytes. Having confirmed its expression in keratinocytes, we tested if VISTA regulates UV-triggered IFN-I production in the skin. Expression analysis of IFN-I stimulated genes in skin biopsies collected prior to and 3h after a single dose of UVB, revealed ~10-fold higher IFN-I score in Vsir−/− vs. B6 skin after UV. RNAseq studies showed DNA sensors, ZBP1, cGAS, and IFI204 were upregulated, while nucleotide excision repair genes, Polɛ and DNA Ligase1, were suppressed in Vsir−/− skin, both at baseline and 3h after UV. Moreover, Polɛ protein levels were lower in Vsir−/− skin by immunoblot. Relevant to its role in keratinocytes, IFN-k protein levels were higher in VISTA-deficient epidermal keratinocytes ex vivo, accompanied by elevated mRNA levels of ZBP1 and IFI204 before and after UV. Together, this study identifies VISTA as a novel regulator of IFN-I production, by modulating DNA damage repair and sensing. The keratinocyte-intrinsic role of VISTA is therapeutically relevant as lupus keratinocytes express high IFN-k and are direct targets of UV-induced DNA damage. 1R21AR079661 – 01, Hitchcock Foundation, DOD lupus award

Towards the controlled enzymatic synthesis of LNA containing oligonucleotides.

Enzymatic, de novo XNA synthesis represents an alternative method for the production of long oligonucleotides containing chemical modifications at distinct locations. While such an approach is currently developed for DNA, controlled enzymatic synthesis of XNA remains at a relative state of infancy. In order to protect the masking groups of 3'-O-modified LNA and DNA nucleotides against removal caused by phosphatase and esterase activities of polymerases, we report the synthesis and biochemical characterization of nucleotides equipped with ether and robust ester moieties. While the resulting ester-modified nucleotides appear to be poor substrates for polymerases, ether-blocked LNA and DNA nucleotides are readily incorporated into DNA. However, removal of the protecting groups and modest incorporation yields represent obstacles for LNA synthesis via this route. On the other hand, we have also shown that the template-independent RNA polymerase PUP represents a valid alternative to the TdT and we have also explored the possibility of using engineered DNA polymerases to increase substrate tolerance for such heavily modified nucleotide analogs.

Purinergic signaling and purine base metabolism at the crossroads between immunity, metabolism, and cancer: A review

AbstractIn addition to its universally known role in transferring genetic material, DNA nucleotides and nucleosides are regarded as the most ancient form of extracellular signaling molecules. This unique signaling pathway was first reported by Dr. Albert Szent‐Györgyi in 1937 and established by Dr. Geoffrey Burnstock in 1972, who coined the term “purinergic signaling.” The significance of purinergic signaling is now recognized in various cellular processes, including immune responses. With an increased understanding of how changes in immunity impact cancer progression, the groundbreaking successes of immunotherapies, and emerging challenges facing patients receiving these treatments, in this review we revisit the history of purinergic signaling, provide a comprehensive summary of its impact on immune cells, and discuss the therapeutic potential of targeting this pathway for cancer treatment.

Pathogenicity of Alternaria Species Causing Tomato Blight in Agro-Ecological Zones of Kirinyaga County in Kenya

Tomato (Lycopersicon esculentum Mill) is a highly consumed and produced vegetable crop with an annual global production of over 185 million tonnes. High consumption and production are due to its nutritional and health benefits. However, tomato production is characterized by inconsistent quality and yields, partly emanating from biotic constraints attributed to fungal foliar diseases. In Kirinyaga County, where tomato production is a significant source of livelihood, knowledge gaps exist regarding the identity of Alternaria species causing tomato blight in different Agro-ecological zones (AEZs) and the susceptibility of locally grown tomato varieties to the Alternaria pathogens. To address these gaps, we conducted a study to determine the characteristics of an Alternaria pathogen isolated from tomato leaves from different AEZs and assess the susceptibility level of some tomato varieties grown in Kirinyaga County. Pathogen characterization was done using phenotypic and molecular approaches, and the susceptibility assay was evaluated by artificial inoculation. The molecular characterization involved BLASTIN analysis of Sanger sequenced DNA nucleotide amplicons, and the use of NCBI databases. The susceptibility assay was carried out in the greenhouse using a completely randomized design. Data on the fungal conidial lengths (µm) and the susceptibility (%) of tomato varieties to fungal isolates were subjected to Analysis of Variance (ANOVA) with significance means separated using LSD at α = 0.05. The morphologies of the pathogens differed significantly (p <0.05). Molecular characterization implicated Alternaria solani, Alternaria cerealis, Alternaria arborescens, and Alternaria alternate in causing tomato blight. Tomato varieties differed in their susceptibility to Alternaria pathogens (p < 0.05). Kilele F1 (73.84%), and Terminator F1 (56.48%) were the most and least susceptible tomato varieties, respectively. Thus, Terminator F1 can serve as a source for the genetic improvement of tomato varieties in the Kirinyaga patho-system. Our study provides valuable information on the identification of Alternaria species causing tomato blight in different AEZs in Kirinyaga County and the susceptibility of locally grown tomato varieties to infection by Alternaria pathogen isolates. The findings can aid in the development of effective disease management strategies and the genetic improvement of tomato varieties in the area.

Low dose acetyl salicylic acid (LDA) mediates epigenetic changes in preeclampsia placental mesenchymal stem cells similar to cells from healthy pregnancy

IntroductionPreeclampsia (PE) affects 2–8% of all pregnancies, and is the leading cause of maternal and fetal morbidity and mortality. We reported on pathophysiological changes in placenta mesenchymal stem cells (P-MSCs) in PE. P-MSCs can be isolated from different layers of the placenta at the interface between the fetus and mother. The ability of MSCs from other sources to be immune licensed as immune suppressor cells indicated that P-MSCs could mitigate fetal rejection. Acetylsalicylic acid (aspirin) is indicated for treating PE. Indeed, low-dose aspirin is recommended to prevent PE in high risk patients. MethodsWe conducted robust computational analyses to study changes in gene expression in P-MSCs from PE and healthy term pregnancies as compared with PE-MSCs treated with low dose acetyl salicylic acid (LDA). Confocal microscopy studied phospho-H2AX levels in P-MSCs. ResultsWe identified changes in >400 genes with LDA, similar to levels of healthy pregnancy. The top canonical pathways that incorporate these genes were linked to DNA repair damage - Basic excision repair (BER), Nucleotide excision repair (NER) and DNA replication. A role for the sumoylation (SUMO) pathway, which could regulate gene expression and protein stabilization was significant although reduced as compared to BER and NER pathways. Labeling for phopho-H2AX indicated no evidence of double strand break in PE P-MSCs. DiscussionThe overlapping of key genes within each pathway suggested a major role for LDA in the epigenetic landscape of PE P-MSCs. Overall, this study showed a new insight into how LDA reset the P-MSCs in PE subjects around the DNA.

Exaptation of Inactivated Host Enzymes for Structural Roles in Orthopoxviruses and Novel Folds of Virus Proteins Revealed by Protein Structure Modeling.

Viruses with large, double-stranded DNA genomes captured the majority of their genes from their hosts at different stages of evolution. The origins of many virus genes are readily detected through significant sequence similarity with cellular homologs. In particular, this is the case for virus enzymes, such as DNA and RNA polymerases or nucleotide kinases, that retain their catalytic activity after capture by an ancestral virus. However, a large fraction of virus genes have no readily detectable cellular homologs, meaning that their origins remain enigmatic. We explored the potential origins of such proteins that are encoded in the genomes of orthopoxviruses, a thoroughly studied virus genus that includes major human pathogens. To this end, we used AlphaFold2 to predict the structures of all 214 proteins that are encoded by orthopoxviruses. Among the proteins of unknown provenance, structure prediction yielded clear indications of origin for 14 of them and validated several inferences that were previously made via sequence analysis. A notable emerging trend is the exaptation of enzymes from cellular organisms for nonenzymatic, structural roles in virus reproduction that is accompanied by the disruption of catalytic sites and by an overall drastic divergence that precludes homology detection at the sequence level. Among the 16 orthopoxvirus proteins that were found to be inactivated enzyme derivatives are the poxvirus replication processivity factor A20, which is an inactivated NAD-dependent DNA ligase; the major core protein A3, which is an inactivated deubiquitinase; F11, which is an inactivated prolyl hydroxylase; and more similar cases. For nearly one-third of the orthopoxvirus virion proteins, no significantly similar structures were identified, suggesting exaptation with subsequent major structural rearrangement that yielded unique protein folds. IMPORTANCE Protein structures are more strongly conserved in evolution than are amino acid sequences. Comparative structural analysis is particularly important for inferring the origins of viral proteins that typically evolve at high rates. We used a powerful protein structure modeling method, namely, AlphaFold2, to model the structures of all orthopoxvirus proteins and compared them to all available protein structures. Multiple cases of recruitment of host enzymes for structural roles in viruses, accompanied by the disruption of catalytic sites, were discovered. However, many viral proteins appear to have evolved unique structural folds.

Abstract 230: Characterizing pathogenic germline variants in a large Chinese lung cancer cohort

Abstract Objective: To characterize germline pathogenic/likely pathogenic variants (GPVs) in Chinese lung cancer patients and analyze the prevalence of the GPVs and their association with lung cancer risk as well as various clinical and genomic features. Methods: A total of 17,904 Chinese lung cancer patients were included in the study. Samples of all 17,904 patients were subjected to next-generation targeted sequencing by using a panel covering more than 400 cancer genes, of which 146 are known cancer predisposition genes. Baseline somatic mutations of 5,460 patients were also obtained by targeted sequencing. Germline variants were called from white blood cells, and the GPVs were determined according to the 2015 ACMG Guideline. The prevalence of the GPVs as well as corresponding pathways in the whole cohort and in various subgroups was compared. The odds ratios of the GPVs for lung cancer risk were estimated by case-control analysis based on the local cohort and the gnomAD East Asia database. Interaction between GPVs and somatic mutations was investigated. Results: Among 17,904 patients, 1,738 patients (9.7%) carried 1,840 GPVs from 87 cancer susceptibility genes. SBDS (1.37%), TSHR (1.20%), BLM (0.62%), BRCA2 (0.62%) and ATM (0.45%) were the top 5 genes with the highest prevalence in the whole cohort. GPVs in thirteen genes including FANCF, KIT, LHCGR, MAP2K2, NRAS, etc. have not been reported in lung cancer patients previously. GPVs in certain genes showed histological variation. GPVs in PTEN, BRCA2, WRN, RAD51C, JAK2 and ERBB2 were more enriched in lung squamous cell carcinoma compared with lung adenocarcinoma (LUAD). At pathway level, homologous recombination repair (HRR, 2.09%), TP53 pathway (0.83%), nucleotide excision repair (NER, 0.7%), Fanconi anemia (FA, 0.6%) and DNA damage sensor (0.53%) ranked the first 5 pathways with the highest overall prevalence. Case-control analysis showed SBDS c.184A>T(p.K62*) had the highest odds ratio for lung cancer risk (OR=14.42, P<0.001), followed by JAK2 c.1849G>T(p.V617F)(OR=10.8, P=0.002) and MUTYH c.55C>T(p.R19*)(OR=9.77, P=0.004). The rate of second hit was 4.74% in 464 patients with germline alterations in tumor suppressor genes. Patients with second hit had a higher percentage with family cancer history (68.2% vs. 35.2%, P=0.011), and tumors with second hit showed higher tumor mutation burden (P=3.2e-05) and chromosome instability score (CIS, P=0.12). PGVs in certain genes were associated with early onset of lung cancer, and co-occurrence analysis showed there existed germline-somatic mutation interaction in lung cancer. Conclusion: Chinese lung cancer patients have a relatively high carry rate of PGVs. PGVs are distributed in multiple pathways and dominated by DNA damage repair associated pathways. The association between identified PGVs and lung cancer risk requires further studies to verify. Citation Format: Xiaoying Wu, Xiaojun Fan, Jiaohui Pang, Hua Bao, Jiani Yin, Haimeng Tang, Xue Wu, Yang Shao. Characterizing pathogenic germline variants in a large Chinese lung cancer cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 230.

Abstract 2610: GNAQ/GNA11 and BAP1 mutant isogenic cell lines engineered by CRISPR/Cas9 gene editing to model ocular melanoma

Abstract Uveal melanoma (UM) is the most frequent intraocular cancer in adults and 50% of patients develop liver metastases for which no treatment is effective. According to The Cancer Genome Atlas, 92% of cases are characterized by early mutations in two genes coding for alpha subunits of G protein coupled receptors (GPCR): G protein subunit alpha q (GNAQ) or 11 (GNA11). These mutations are mostly found in the codon Q209 causing a constitutive activation of different signaling pathways linked to cellular proliferation such as MAPK and PKC. Furthermore, a bi-allelic inactivation of the BRCA1 associated protein-1 (BAP1) is found in 83% of metastatic UM and causes a loss of function of this protein. Our hypothesis is that the genes essential for the survival of mutant UM cells will be ideal therapeutic targets. Using CRISPR/Cas9 technology, we engineered GNAQ/11 and BAP1 mutant isogenic cell lines derived from wildtype UM cells or choroidal melanocytes (CM) to better understand the molecular mechanisms involved in UM development. We have designed three ribonucleoprotein complexes (RNPs) composed of single-guide RNA (sgRNA) and Streptococcus pyogenes Cas9 (spCas9) specific to the GNAQ/11 and BAP1 genes. This allowed to target the DNA sequence on the exon 5 of these proteins to induce a double-strand break. To introduce the desired mutations Q209L in GNAQ/11 or C91G in BAP1, we have also designed DNA templates called single-strand oligo DNA nucleotides (ssODNs) that were co-transfected with respective RNPs by electroporation into UM cells (Mel285) or CM and favorized the homologous recombination (HR) during DNA repair to knock-in these mutations. After a few days, we genotyped the clones by Sanger sequencing and expanded those of interest into cell lines. We obtained an electroporation efficiency of 50% for CM and 70% for Mel285 using an eGFP plasmid. The first CRISPR transfections showed a mean edition of 20-60% of targeted regions with 4% HR. To increase our percentage of HR, we have also used the Prime Editing method; nine plasmids per mutation have been designed and constructed. The first transfection tests in HEK293T cells are currently underway. We obtained three KO cell lines: Mel285GNAQ-KO, Mel285GNA11-KO and Mel285BAP1-KO. We saw no morphologic changes. A decrease in the proliferation and metabolic activity was observed in Mel285GNAQ-KO and Mel285BAP1-KO cell lines in comparison to the wildtype cells. Furthermore, an increase of phosphorylated ATF2/7 was noted in Mel285GNAQ-KO and Mel285GNA11-KO cell lines by western blotting. A better understanding of the altered pathways in our GNAQ/11 or BAP1 mutant isogenic cell lines will help to identify new drugs targeting specifically metastatic UM cells. Citation Format: Aurélie Fuentes-Rodriguez, Andrew Mitchell, Joël Rousseau, Jacques P. Tremblay, Solange Landreville. GNAQ/GNA11 and BAP1 mutant isogenic cell lines engineered by CRISPR/Cas9 gene editing to model ocular melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2610.

Mitochondrial carnitine palmitoyltransferase-II dysfunction: A possible novel mechanism for nonalcoholic fatty liver disease in hepatocarcinogenesis.

Nonalcoholic fatty liver disease (NAFLD) or metabolic-associated fatty liver disease has been characterized by the lipid accumulation with injury of hepatocytes and has become one of the most common chronic liver diseases in the world. The complex mechanisms of NAFLD formation are still under identification. Carnitine palmitoyltransferase-II (CPT-II) on inner mitochondrial membrane (IMM) regulates long chain fatty acid β-oxidation, and its abnormality has had more and more attention paid to it by basic and clinical research in NAFLD. The sequences of its peptide chain and DNA nucleotides have been identified, and the catalytic activity of CPT-II is affected on its gene mutations, deficiency, enzymatic thermal instability, circulating carnitine level and so on. Recently, the CPT-II dysfunction has been discovered in models of liver lipid accumulation. Meanwhile, the malignant transformation of hepatocyte-related CD44+ stem T cell activation, high levels of tumor-related biomarkers (AFP, GPC3) and abnormal activation of Wnt3a expression as a key signal molecule of the Wnt/β-catenin pathway run parallel to the alterations of hepatocyte pathology. This review focuses on some of the progress of CPT-II inactivity on IMM with liver fatty accumulation as a possible novel pathogenesis for NAFLD in hepatocarcinogenesis.

Phylogenic study of toxoplasmosis in aborted women in Al- Diwnaniya province

The current study included the detection of Toxoplasma gondii from aborted women attented to the Maternity and Childhood Teaching Hospital to detected the rate of abortion due to toxoplasmosis using PCR technique, the samples which was used placenta and fetus, this study also identify the differentiation between it by phylogenic with SAG3 gene.
 A total of 60 ( placenta and fetus) samples were collected randomly from aborted women who received to the Maternity and Childhood Teaching Hospital.
 The present study include molecular method to detected small subunit ribosomal RNA ( ssRNA) using Nested Polymerase Chain Reaction ( nPCR) ,the results of the nPCR for ssRNA gene, the result showed that the rate of toxoplasmosis in the aborted women was Positive 60%. (There is a statistically significant between species in P <0.05).
 The present study also detected for Surface Antigen (SAG3) gene to conform the nPCP result and to send the samples for sequences.
 The SAGA gene showed 100% positive results according to the ssRNA gene .
 T.gondii isolates surface Ag SAG3 gene with NCBI BLAST Toxoplasma gondii isolates, and the DNA nucleotides sequencing analysis of surface Ag SAG3 complete gene was show clear gentic variation between isolates from different host. 

Broad-host-range lytic Erwinia phage Key with exopolysaccharide degrading activity

In this study, the genome of the lytic broad-host-range phage Key infecting Erwinia amylovora, Erwinia horticola, and Pantoea agglomerans strains was characterized. Key phage has a 115,651 bp long double-stranded DNA genome with the G + C ratio of 39.03%, encoding 182 proteins and 27 tRNA genes. The majority (69%) of predicted coding sequences (CDSs) encode proteins with unknown functions. The protein products of 57 annotated genes were found to have probable functions in nucleotide metabolism, DNA replication, recombination, repair, and packaging, virion morphogenesis, phage-host interaction and lysis. Furthermore, the product of gene 141 shared amino acid sequence similarity and conserved domain architecture with the exopolysaccharide (EPS) degrading proteins of Erwinia and Pantoea infecting phages as well as bacterial EPS biosynthesis proteins. Due to the genome synteny and similarity to the proteins of T5-related phages, phage Key, together with its closest relative, Pantoea phage AAS21, was suggested to represent a novel genus within the Demerecviridae family, for which we tentatively propose the name “Keyvirus”.

Multimolecular characteristics and role of BRCA1 interacting protein C-terminal helicase 1 (BRIP1) in human tumors: a pan-cancer analysis

BackgroundThe aberrant expression of BRIP1 was associated with several cancers; however, the panoramic picture of BRIP1 in human tumors remains unclear. This study aims to explore the pan-cancerous picture of the expression of BRIP1 across 33 human cancers.MethodsBased on the data from TCGA and GTEx, a series of bioinformatic analyses were applied to systematically explore the genetic landscape and biologic function of BRIP1 in 33 human tumors.ResultsWe observed prognosis-related differential BRIP1 expressions between various carcinomas and the corresponding normal tissues. “Basal transcription factors,” “homologous recombination,” “nucleotide excision repair,” and DNA metabolism pathways may play a role in the functional mechanisms of BRIP1. Patients with uterine corpus endometrial carcinoma presented with the highest alteration frequency of BRIP1 (nearly 10%). Single-nucleotide and copy number variations of BRIP1 were noticed in multiple cancers, and the expression of BRIP1 is significantly regulated by copy number variation in breast invasive carcinoma and lung squamous cell carcinoma. BRIP1 expression is negatively correlated with the DNA methylation levels in many tumors and is associated with the activation of apoptosis, cell cycle, DNA damage response, and inhibition of hormone ER and RNS/MARK signaling pathways. Moreover, a positive correlation was observed between BRIP1 expression and the immune infiltration levels of cancer-associated fibroblasts and CD8+ T cells in lung adenocarcinoma.ConclusionOur pan-cancer analysis of BRIP1 provides a valuable resource for understanding the multimolecular characteristics and biological function of BRIP1 across human cancers.

Bayesian Pyramids: identifiable multilayer discrete latent structure models for discrete data

Abstract High-dimensional categorical data are routinely collected in biomedical and social sciences. It is of great importance to build interpretable parsimonious models that perform dimension reduction and uncover meaningful latent structures from such discrete data. Identifiability is a fundamental requirement for valid modeling and inference in such scenarios, yet is challenging to address when there are complex latent structures. In this article, we propose a class of identifiable multilayer (potentially deep) discrete latent structure models for discrete data, termed Bayesian Pyramids. We establish the identifiability of Bayesian Pyramids by developing novel transparent conditions on the pyramid-shaped deep latent directed graph. The proposed identifiability conditions can ensure Bayesian posterior consistency under suitable priors. As an illustration, we consider the two-latent-layer model and propose a Bayesian shrinkage estimation approach. Simulation results for this model corroborate the identifiability and estimatability of model parameters. Applications of the methodology to DNA nucleotide sequence data uncover useful discrete latent features that are highly predictive of sequence types. The proposed framework provides a recipe for interpretable unsupervised learning of discrete data and can be a useful alternative to popular machine learning methods.

Mitochondrial DNA Polymorphism in HV1 and HV2 Regions and 12S rDNA in Perimenopausal Hypertensive Women.

Estrogens enhance cellular mitochondrial activity. The diminution of female hormones during menopause may have an effect on the mitochondrial genome and the expression of mitochondrial proteins. Hence, oxidative stress and the pro-inflammatory state contribute to the formation of systemic illnesses including arterial hypertension (AH). This study aimed to determine the types and frequency of mutations in the mitochondrial DNA (mtDNA) nucleotide sequence in the hypervariable regions 1 and 2 (HV1 and HV2) and the 12S RNA coding sequence of the D-loop in postmenopausal women with hypertension. In our study, 100 women were investigated, 53 of whom were postmenopausal and 47 of whom were premenopausal (53.9 ± 3.7 years vs. 47.7 ± 4.2 years, respectively). Of those studied, 35 premenopausal and 40 postmenopausal women were diagnosed with AH. A medical checkup with 24 h monitoring of blood pressure (RR) and heart rate was undertaken (HR). The polymorphism of the D-loop and 12S rDNA region of mtDNA was examined. Changes in the nucleotide sequence of mtDNA were observed in 23% of the group of 100 women. The changes were identified in 91.3% of HV1 and HV2 regions, 60.9% of HV1 segments, 47.5% of HV2 regions, and 43.5% of 12S rDNA regions. The frequency of nucleotide sequence alterations in mtDNA was substantially higher in postmenopausal women (34%) than in premenopausal women (10.6%), p = 0.016. A higher frequency of changes in HV1 + HV2 sections in postmenopausal women (30.2%) compared to the premenopausal group (10.6%) was detected, p = 0.011. Only postmenopausal women were found to have modifications to the HV2 segment and the 12S rDNA region. After menopause, polymorphism in the mtDNA region was substantially more frequent in women with arterial hypertension than before menopause (p = 0.030; 37.5% vs. 11.5%). Comparable findings were observed in the HV2 and HV1 regions of the AH group (35% vs. 11.5%), p = 0.015, in the HV1 segment (25% vs. 11.5%), p = 0.529, and in the HV2 segment, 12S rDNA (25% vs. 0%). More than 80% of all changes in nucleotide sequence were homoplasmic. The mtDNA polymorphisms of the nucleotide sequence in the HV1 and HV2 regions, the HV2 region alone, and the 12S RNA coding sequence were associated with estrogen deficiency and a more severe course of arterial hypertension, accompanied by symptoms of adrenergic stimulation.

Sperm DNA damage: The possible link between obesity and male infertility, an update of the current literature.

Obesity prevalence worldwide is increasing significantly. Whilst maternal obesity has clear detrimental impacts on fertility, pregnancy and foetal outcomes, more recently there has been an increasing focus on the role of paternal obesity in human fertility. Recent meta-analyses have indicated that obesity in men negatively affects basic sperm parameters such as sperm count, concentration and motility, increases the incidence of infertility and reduces the chances of conception. Sperm DNA damage, typically characterised by DNA strand breaks and oxidation of DNA nucleotides, is a specialised marker of sperm quality that has been independently associated with recurrent miscarriage, reduced assisted reproduction success and increased mutational loads in subsequent offspring. Whilst, there are still conflicting data in humans as to the association of obesity in men with sperm DNA damage, evidence from rodent models is clear, indicating that male obesity increases sperm DNA damage. Human data are often conflicting because of the large heterogeneity amongst studies, the use of body mass index as the indicator of obesity and the methods used for detection of sperm DNA damage. Furthermore, comorbidities of obesity (i.e., heat stress, adipokines, insulin resistance, changes in lipids, hypogonadism and obstructive sleep apnoea) are also independently associated with increased sperm DNA damage that is not always modified in men with obesity, and as such may provide a causative link to the discrepancies amongst human studies. In this review, we provide an update on the literature regarding the associations between obesity in men and fertility, basic sperm parameters and sperm DNA damage. We further discuss potential reasons for the discrepancies in the literature and outline possible direct and indirect mechanisms of increased sperm DNA damage resulting from obesity. Finally, we summarise intergenerational obesity through the paternal linage and how sperm DNA damage may contribute to the transmission.

Facial clues to the photosensitive trichothiodystrophy phenotype in childhood.

Among genodermatoses, trichothiodystrophies (TTDs) are a rare genetically heterogeneous group of syndromic conditions, presenting with skin, hair, and nail abnormalities. An extra-cutaneous involvement (craniofacial district and neurodevelopment) can be also a part of the clinical picture. The presence of photosensitivity describes three forms of TTDs: MIM#601675 (TTD1), MIM#616390 (TTD2) and MIM#616395 (TTD3), that are caused by variants afflicting some components of the DNA Nucleotide Excision Repair (NER) complex and with more marked clinical consequences. In the present research, 24 frontal images of paediatric patients with photosensitive TTDs suitable for facial analysis through the next-generation phenotyping (NGP) technology were obtained from the medical literature. The pictures were compared to age and sex-matched to unaffected controls using 2 distinct deep-learning algorithms: DeepGestalt and GestaltMatcher (Face2Gene, FDNA Inc., USA). To give further support to the observed results, a careful clinical revision was undertaken for each facial feature in paediatric patients with TTD1 or TTD2 or TTD3. Interestingly, a distinctive facial phenotype emerged by the NGP analysis delineating a specific craniofacial dysmorphic spectrum. In addition, we tabulated every single detail within the observed cohort. The novelty of the present research includes the facial characterization in children with the photosensitive types of TTDs through the 2 different algorithms. This result can become additional criteria for early diagnosis, and for subsequent targeted molecular investigations as well as a possible tailored multidisciplinary personalized management.

Research and Therapeutic Approaches in Stem Cell Genome Editing by CRISPR Toolkit.

The most widely used genome editing toolkit is CRISPR (clustered regularly interspaced short palindromic repeats). It provides the possibility of replacing and modifying DNA and RNA nucleotides. Furthermore, with advancements in biological technology, inhibition and activation of the transcription of specific gene(s) has become possible. Bioinformatics tools that target the evolution of CRISPR-associated protein 9 (Cas9) turn this protein into a vehicle that is specific for a DNA or RNA region with single guide RNA (sgRNA). This toolkit could be used by researchers to investigate the function of stem cell gene(s). Here, in this review article, we cover recent developments and applications of this technique in stem cells for research and clinical purposes and discuss different CRISPR/Cas technologies for knock-out, knock-in, activation, or inhibition of gene expression. Additionally, a comparison of several deliveries and off-target detecting strategies is discussed.

Multiple transitions between realms shape relict lineages of Proteus cave salamanders.

In comparison to biodiversity on Earth's surface, subterranean biodiversity has largely remained concealed. The olm (Proteus anguinus) is one of the most enigmatic extant cave inhabitants, and until now little was known regarding its genetic structure and evolutionary history. Olms inhabit subterranean waters throughout the Dinaric Karst of the western Balkans, with a seemingly uniform phenotypic appearance of cave-specialized traits: an elongate body, snout and limbs, degenerated eyes and loss of pigmentation ("white olm"). Only a single small region in southeastern Slovenia harbours olms with a phenotype typical of surface animals: pigmented skin, eyes, a blunt snout and short limbs ("black olm"). We used a combination of mitochondrial DNA and genome-wide single nucleotide polymorphism data to investigate the molecular diversity, evolutionary history and biogeography of olms along the Dinaric Karst. We found nine deeply divergent species-level lineages that separated between 17 and 4 million years ago, while molecular diversity within lineages was low. We detected no signal of recent admixture between lineages and only limited historical gene flow. Biogeographically, the contemporaneous distribution of lineages mostly mirrors hydrologically separated subterranean environments, while the historical separation of olm lineages follows microtectonic and climatic changes in the area. The reconstructed phylogeny suggests at least four independent transitions to the cave phenotype. Two of the species-level lineages have miniscule ranges and may represent Europe's rarest amphibians. Their rarity and the decline in other lineages call for protection of their subterranean habitats.

Sequence‐dependent clustering properties of nucleotides fragments in an ionic solution

AbstractThe chemical nature of the DNA bases is an important factor in sequence‐mediated association of DNA molecules. Nucleotides are the fundamental DNA elements and the base identity impacts the molecular properties of nucleotide fragments. It is interesting to study the fundamental nature of nucleotides in DNA, on the basis of base‐specific interactions, association, and modes of standard atomic or molecular interactions. With all‐atom molecular dynamics simulations of model dinucleotide and tetranucleotide systems having single‐stranded dinucleotide or tetranucleotide fragments of varying sequences, we show how the base identity and interactions between the different bases as well as water may affect the clustering properties of nucleotides fragments in an ionic solution. Sequence‐dependent differential interactions between the nucleotide fragments, ionic concentration, and elevated temperature are found to influence the clustering properties and dynamics of association. Well‐known epigenetic modification of DNA, that is, cytosine methylation also promotes dinucleotide clustering in solution. These observations point to one possible chemical nature of the DNA bases, as well as the importance of the base pairing, base stacking, and ionic interactions in DNA structure formation, and DNA sequence‐mediated association. Sequence‐ and the ionic environment‐mediated self‐association properties of the dinucleotides indicate its great potential to develop biological nanomaterials for desired applications.

PH dynamics regulates transcription factor-DNA binding.

Intracellular pH (pHi) dynamics regulates myriad cell behaviors, and we previously identified pH-sensitive proteins regulating cell migration, cytoskeleton and adhesion remodeling, and transformation through pH-dependent effects on protein-protein and protein-phospholipid binding. Whether pH regulates protein-nucleotide binding for established effects of pHi dynamics on gene expression, however, remains unknown. We identified at least 65 transcription factors in diverse families that have a conserved histidine, which in available structures forms hydrogen bonds with DNA nucleotides. With the ability of histidine to titrate in the cellular pH range, electrostatic principles of protein-nucleotide hydrogen bonds, we predicted that transcription factors with histidine-nucleotide hydrogen bonds could have pH regulated DNA binding selectivity. All FOX family transcription factors contain a conserved histidine in their DNA-binding domain (DBD) that in available structures forms hydrogen bonds with nucleotides. We confirmed pH regulates DNA binding of FOXM1 and FOXC2 DBDs using fluorescence anisotropy and a canonical FkhP DNA motif for binding to a thymine, with higher affinity at pH 7 compared with 7.5. This finding is consistent with thymine being a hydrogen bond acceptor and binding a protonated histidine. We confirmed the significance of histidine with a mutant FOXC2-His122K, with lysine mimicking a protonated residue, that had high affinity but pH-independent binding and a mutant FOXC2-H122N recurring in cancers that had low affinity pH-independent binding. We also confirmed pHi regulates FOXC2 activity in cells. Using MDA-MB-436 cancer cells with a high pHi of 7.7 and a luciferase assay we found increased transcriptional activity for an FkhP motif when pHi was lowered to 7.4 with EIPA, an inhibitor of the proton-extruding NHE1. Our data identify pH dynamics as a previously unrecognized regulator of DNA-binding selectivity for FOXM1 and FOXC2 with broad significance for transcription factors in diverse families with conserved histidines in the DBD.