AnoPrimer: Primer Design in malaria vectors informed by range-wide genomic variation.

The major malaria mosquitoes, Anopheles gambiae s.l and Anopheles funestus, are some of the most studied organisms in medical research and also some of the most genetically diverse. When designing polymerase chain reaction (PCR) or hybridisation-based molecular assays, reliable primer and probe design is crucial. However, single nucleotide polymorphisms (SNPs) in primer binding sites can prevent primer binding, leading to null alleles, or bind suboptimally, leading to preferential amplification of specific alleles. Given the extreme genetic diversity of Anopheles mosquitoes, researchers need to consider this genetic variation when designing primers and probes to avoid amplification problems. In this note, we present a Python package, AnoPrimer, which exploits the Ag1000G and Af1000 datasets and allows users to rapidly design primers in An. gambiae or An. funestus, whilst summarising genetic variation in the primer binding sites and visualising the position of primer pairs. AnoPrimer allows the design of both genomic DNA and cDNA primers and hybridisation probes. By coupling this Python package with Google Colaboratory, AnoPrimer is an open and accessible platform for primer and probe design, hosted in the cloud for free. AnoPrimer is available here https://github.com/sanjaynagi/AnoPrimer and we hope it will be a useful resource for the community to design probe and primer sets that can be reliably deployed across the An. gambiae and funestus species ranges.

The majority of molecular biology applications require synthetic DNA sequences called primers, which bind to DNA and allow us to amplify specific stretches of DNA which we are interested in. Unfortunately, when mutations occur at primer binding sites, primers can fail to bind completely, or even worse, amplify differentially depending on the mutation present. Mutations can therefore bias molecular assays with often undetected effects. This is a particular problem in malaria mosquitoes, as they are some of the most genetically diverse species on earth. We present a user-friendly software tool, AnoPrimer, which allows users to design primers for molecular biology in malaria mosquitoes. AnoPrimer integrates high-quality whole-genome sequence data from the cloud, and creates clear, interactive visualisations, enabling users to avoid mutations that occur in wild malaria mosquitoes. By avoiding these mutations, we can ensure the design of reliable primers which result in robust molecular assays for research into malaria vectors.

Mitochondrial translation is the primary determinant of secondary mitochondrial complex I deficiencies.

Individual complexes of the mitochondrial oxidative phosphorylation system (OXPHOS) are not linked solely by their function; they also share dependencies at the maintenance/assembly level, where one complex depends on the presence of a different individual complex. Despite the relevance of this "interdependence" behavior for mitochondrial diseases, its true nature remains elusive. To understand the mechanism that can explain this phenomenon, we examined the consequences of the aberration of different OXPHOS complexes in human cells. We demonstrate here that the complete disruption of each of the OXPHOS complexes resulted in a decrease in the complex I (cI) level and that the major reason for this is linked to the downregulation of mitochondrial ribosomal proteins. We conclude that the secondary cI defect is due to mitochondrial protein synthesis attenuation, while the responsible signaling pathways could differ based on the origin of the OXPHOS defect.

FXN targeting induces cell death in ovarian cancer stem-like cells through PRDX3-Mediated oxidative stress.

Ovarian cancer stem cells (OCSCs) significantly impact the prognosis, chemoresistance, and treatment outcomes in OC. While ferroptosis has been proven effective against OCSCs, the intricate relationship between ferroptosis and OCSCs remains incompletely understood. Here, we enriched ovarian cancer stem-like cells (OCSLCs) through mammosphere culture, as an OCSC model. OCSLCs displayed heightened ferroptosis susceptibility, correlating with elevated FXN levels compared to non-stem OC cells. FXN has recently emerged as a potential regulator in ferroptosis. FXN knockdown diminished stemness marker nanog, sphere-forming ability, increased reactive oxygen species (ROS) generation, and attenuated OCSLCs viability. FXN overexpression exacerbated ferroptosis resistance and reduced RSL3-induced cell death. FXN knockdown impeded OCSLC xenograft tumor growth and exacerbated the degeneration of peroxiredoxin 3 (PRDX3), a mitochondrial antioxidant protein participates in oxidative stress. Thus, elevated FXN in OCSLCs suppresses ROS accumulation, fostering ferroptosis resistance, and regulates the antioxidant protein PRDX3. FXN emerges as a potential therapeutic target for OC.

Astroglia proliferate upon the biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase.

Astroglia play crucial neuroprotective roles by internalizing pathogenic aggregates and facilitating their degradation. Here, we show that α-SYN protofibril-induced organelle toxicities and reactive oxygen species (ROS) cause premature cellular senescence in astrocytes and astrocyte-derived cancer cells, resulting in a transient increase in the biogenesis of tunneling nanotubes (TNTs). TNT-biogenesis and TNT-mediated cell-to-cell transfer lead to clearance of α-SYN-induced organelle toxicities, reduction in cellular ROS levels, and reversal of cellular senescence. Enhanced cell proliferation is seen in the post-recovered cells after recovering from α-SYN-induced organelle toxicities. Further, we show that α-SYN-induced senescence promotes the transient localization of focal adhesion kinase (FAK) in the nucleus. FAK-mediated regulation of Rho-associated kinases plays a significant role in the biogenesis of TNTs and their subsequent proliferation. Our study emphasizes that TNT biogenesis has a potential role in the clearance of α-SYN-induced cellular toxicities, the consequences of which cause enhanced proliferation in the post-recovered astroglia cells.

A high-throughput screening identifies MCM chromatin loading inhibitors targeting cells with increased replication origins.

Replication origin assembly is a pivotal step in chromosomal DNA replication. In this process, the ORC complex binds DNA and, together with the CDC6 and CDT1, promotes the loading of the MCM helicase. Chemicals targeting origin assembly might be useful to sensitize highly proliferative cancer cells. However, identifying such compounds is challenging due to the multistage nature of this process. Here, using Xenopus laevis egg extract we set up a high-throughput screening to isolate MCM chromatin loading inhibitors, which led to the identification of NSC-95397 as a powerful inhibitor of replication origin assembly that targets CDC6 protein and promotes its degradation. Using systems developed to test selective drug-induced lethality we show that NSC-95397 triggers cell death both in human cells and Xenopus embryos that have higher proliferative ability. These findings demonstrate the effectiveness of molecules disrupting DNA replication processes in targeting hyperproliferating cells, highlighting their potential as anti-cancer molecules.

Usefulness of endoscopic ultrasound with bronchoscope-guided fine-needle aspiration for next-generation sequencing in patients with non-small cell lung cancer: A comparison with other bronchoscopic techniques.

BACKGROUND: Next-generation sequencing (NGS) is essential in treating advanced lung cancer. However, the effectiveness of endoscopic ultrasound with bronchoscope-guided fine-needle aspiration (EUS-B-FNA) in NGS remains unclear. This study examined the usefulness of EUS-B-FNA in lung cancer NGS cases where EUS-B-FNA was performed for specimen submission in a nationwide genomic screening platform (LC-SCRUM-Asia) and compared specimens collected using other bronchoscopy methods (endobronchial ultrasound-guided transbronchial needle aspiration [EBUS-TBNA] and EBUS-guided transbronchial biopsy with a guide sheath [EBUS-GS-TBB]) during the same period. METHODS: We retrospectively compared the NGS success rates of NGS, DNA and RNA yields for EUS-B-FNA, EBUS-TBNA, and EBUS-GS-TBB from the records of the patients recruited for the Lung Cancer Genomic Screening Project for Individualized Medicine (LC-SCRUM)-Asia. RESULTS: Fifty-one patients were enrolled, and the NGS success rates were comparable for samples obtained by EUS-B-FNA, EBUS-TBNA, and EBUS-GS-TBB (100%, 90.9%, and 81.0%, respectively). Genetic alterations were detected in 73.7%, 90.9%, and 85.7% of patients, respectively, with druggable genetic alterations found in 31.6%, 72.7%, and 61.9% of patients, respectively. The DNA and RNA yields were significantly higher in EUS-B-FNA samples than in EBUS-GS-TBB samples (50.4 (interquartile range (IR): 15.45-72.35) ng/µl and 33.9 (IR: 9-76.8) ng/µl from EUS-B-FNA, and 3.3 (IR: 1.4-7.1) ng/µl and 15.1 (IR: 8.3-31.5) ng/µl from EBUS-GS-TBB, respectively, p < 0.05). CONCLUSION: EUS-B-FNA emerges as a promising bronchoscopic method for obtaining adequate samples for NGS in advanced lung cancer cases.

Conserved role of FOXC1 in TNBC is parallel to FOXA1 in ER+ breast cancer.

Triple-negative breast cancer (TNBC) is characterized by lack of the estrogen (ER) receptor, progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2), and standard receptor-targeted therapies are ineffective. FOXC1, a transcription factor aberrantly overexpressed in many cancers, drives growth, metastasis, and stem-cell-like properties in TNBC. However, the molecular function of FOXC1 is unknown, partly due to heterogeneity of TNBC. Here, we show that although FOXC1 regulates many cancer hallmarks in TNBC, its function is varied in different cell lines, highlighted by the differential response to CDK4/6 inhibitors upon FOXC1 loss. Despite this functional heterogeneity, we show that FOXC1 regulates key oncogenes and tumor suppressors and identify a set of core FOXC1 peaks conserved across TNBC cell lines. We identify the ER-associated and drug-targetable nuclear receptor NR2F2 as a cofactor of FOXC1. Finally, we show that core FOXC1 targets in TNBC are regulated in parallel by the pioneer factor FOXA1 and the nuclear receptor NR2F2 in ER + breast cancer.

Multi-omic analysis reveals VEGFR2, PI3K, and JNK mediate the small molecule induction of human iPSC-derived cardiomyocyte proliferation.

Mammalian hearts lose their regenerative potential shortly after birth. Stimulating the proliferation of preexisting cardiomyocytes is a potential therapeutic strategy for cardiac damage. In a previous study, we identified 30 compounds that induced the bona-fide proliferation of human iPSC-derived cardiomyocytes (hiPSC-CM). Here, we selected five active compounds with diverse targets, including ALK5 and CB1R, and performed multi-omic analyses to identify common mechanisms mediating the cell cycle progression of hiPSC-CM. Transcriptome profiling revealed the top enriched pathways for all compounds including cell cycle, DNA repair, and kinesin pathways. Functional proteomic arrays found that the compounds collectively activated multiple receptor tyrosine kinases including ErbB2, IGF1R, and VEGFR2. Network analysis integrating common transcriptomic and proteomic signatures predicted that MAPK/PI3K pathways mediated compound responses. Furthermore, VEGFR2 negatively regulated endoreplication, enabling the completion of cell division. Thus, in this study, we applied high-content imaging and molecular profiling to establish mechanisms linking pro-proliferative agents to mechanisms of cardiomyocyte cell cycling.

Genetic architecture of long-distance migration and population genomics of the endangered Japanese eel.

The Japanese eel (Anguilla japonica), a flagship anguillid species for conservation, is known for its long-distance-oriented migration. However, our understanding of the genetic architecture underlying long-distance migration and population genomic characteristics of A. japonica is still limited. Here, we generated a high-quality chromosome-level genome assembly and conducted whole-genome resequencing of 218 individuals to explore these aspects. Strong signals of selection were found on genes involved in long-distance aerobic exercise and navigation, which might be associated with evolutionary adaptation to long-distance migrations. Low genetic diversity was detected, which might result from genetic drift associated with demographic declines. Both mitochondrial and nuclear genomic datasets supported the existence of a single panmictic population for Japanese eel, despite signals of single-generation selection. Candidate genes for local selection involved in functions like development and circadian rhythm. The findings can provide insights to adaptative evolution to long-distance migration and inform conservation efforts for A. japonica.

Validation of heat-inducible Ixodes scapularis HSP70 and tick-specific 3xP3 promoters in ISE6 cells.

Ixodes scapularis is an important vector of many pathogens, including the causative agent of Lyme disease. The gene function studies in I. scapularis and other ticks are hampered by the lack of genetic tools, including an inducible promoter for temporal control over transgene-encoding protein or double-stranded RNA. We characterized an intergenic sequence upstream of a heat shock protein 70 (HSP70) gene that can drive Renilla luciferase and mCherry expression in the I. scapularis cell line ISE6 (IsHSP70). In another construct, we replaced the Drosophila melanogaster minimal HSP70 promoter of the 3xP3 promoter with a minimal portion of IsHSP70 promoter and generated an I. scapularis-specific 3xP3 (Is3xP3) promoter. Both IsHSP70 and Is3xP3 have a heat-inducible expression of mCherry fluorescence in ISE6 cells with an approximately 10-fold increase in the percentage of fluorescent cells upon 2 h heat shock. These promoters described will be valuable tools for gene function studies.

RPL22 is a tumor suppressor in MSI-high cancers and a splicing regulator of MDM4.

Microsatellite instability-high (MSI-H) tumors are malignant tumors that, despite harboring a high mutational burden, often have intact TP53. One of the most frequent mutations in MSI-H tumors is a frameshift mutation in RPL22, a ribosomal protein. Here, we identified RPL22 as a modulator of MDM4 splicing through an alternative splicing switch in exon 6. RPL22 loss increases MDM4 exon 6 inclusion and cell proliferation and augments resistance to the MDM inhibitor Nutlin-3a. RPL22 represses the expression of its paralog, RPL22L1, by mediating the splicing of a cryptic exon corresponding to a truncated transcript. Therefore, damaging mutations in RPL22 drive oncogenic MDM4 induction and reveal a common splicing circuit in MSI-H tumors that may inform therapeutic targeting of the MDM4-p53 axis and oncogenic RPL22L1 induction.

Androgen receptor-induced molecules and androgen contribute synergistically to male-predominance of hepatocellular carcinoma.

We aimed to clarify the mechanisms of male predominance of hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC). Androgen receptor (AR) facilitates HCC cell growth, which was augmented by androgen (dihydrotestosterone [DHT]) and attenuated by anti-androgen (flutamide). AR upregulated the expressions of BIRC7, IGFBP3, and NTSR1 via increasing their promoter activities, which were enhanced by DHT. Wild-type HBV X (WT-HBx) upregulated AR transcription, which depended on DHT; whereas the effect of C-terminal carboxy-truncated HBx on AR transcription was independent of DHT. BIRC7, IGFBP3, and NTSR1 increased the growth of HCC. High expression of BIRC7 and NTSR1 contributes to poor HCC outcomes in male patients, but not in female patients. Downregulation of NTSR1 inhibits tumor growth in male mice rather than in female mice. Conclusively, AR promotes HCC at least partially via upregulating BIRC7, IGFBP3, and NTSR1, which is enhanced by androgen and HBx. BIRC7 and NTSR1 facilitate HCC progression in a male-predominant manner.

Reversion of pathogenic BRCA1 L1780P mutation confers resistance to PARP and ATM inhibitor in breast cancer.

This study investigates the molecular characteristics and therapeutic implications of the BRCA1 L1780P mutation, a rare variant prevalent among Korean hereditary breast cancer patients. Using patient-derived xenograft (PDX) models and cell lines (PDX-derived cell line) from carriers, sequencing analyses revealed loss of heterozygosity (LOH) at the BRCA1 locus, with one patient losing the wild-type allele and the other mutated allele. This reversion mutation may cf. resistance to homologous recombination deficiency (HRD)-targeting drugs such as PARP inhibitors (PARPi) and ATM inhibitors (ATMi). Although HRDetect and CHORD analyses confirmed a strong association between the L1780P mutation and HRD, effective initially, drug resistance developed in cases with reversion mutations. These findings underscore the complexity of using HRD prediction in personalized treatment strategies for breast cancer patients with BRCA1/2 mutations, as resistance may arise in reversion cases despite high HRD scores.

Amphiregulin orchestrates the paracrine immune-suppressive function of amniotic-derived cells through its interplay with COX-2/PGE2/EP4 axis.

The paracrine crosstalk between amniotic-derived membranes (AMs)/epithelial cells (AECs) and immune cells is pivotal in tissue healing following inflammation. Despite evidence collected to date, gaps in understanding the underlying molecular mechanisms have hindered clinical applications. The present study represents a significant step forward demonstrating that amphiregulin (AREG) orchestrates the native immunomodulatory functions of amniotic derivatives via the COX-2/PGE2/EP4 axis. The results highlight the immunosuppressive efficacy of PGE2-dependent AREG release, dampening PBMCs' activation, and NFAT pathway in Jurkat reporter cells via TGF-ß signaling. Moreover, AREG emerges as a key protein mediator by attenuating acute inflammatory response in Tg(lysC:DsRed2) zebrafish larvae. Notably, the interplay of diverse COX-2/PGE2 pathway activators enables AM/AEC to adapt rapidly to external stimuli (LPS and/or stretching) through a responsive positive feedback loop on the AREG/EGFR axis. These findings offer valuable insights for developing innovative cell-free therapies leveraging the potential of amniotic derivatives in immune-mediated diseases and regenerative medicine.

STED super-resolution microscopy unveils the dynamics of Atg30 on yeast Pex3-labeled peroxisomes.

Peroxisomes are dynamic organelles with important metabolic functions. Yeast Pex3 is a multifunctional membrane protein aiding in peroxisomal biogenesis, inheritance, and degradation (pexophagy), by interacting with process-specific factors. Using multicolor (live-cell) stimulated emission depletion (STED) nanoscopy, we studied the localization of Pex3 and its binding partners in Hansenula polymorpha. Unlike confocal microscopy, STED allows resolving the membrane of tiny peroxisomes, enabling accurate measurements of the size of all Pex3-labeled peroxisomes. We localized Pex3 and its binding partners at peroxisome-repressing and -inducing conditions and during pexophagy. In-depth quantitative analysis of Pex3 and pexophagy receptor Atg30 showed dynamic changes in their (co)localization. One remarkable response of Atg30 was the shift in position from being sandwiched between clustered peroxisomes at proliferation conditions, to the cytosolically exposed parts of peroxisome clusters upon pexophagy induction. Summarizing, we show that STED allows characterizing dynamics of the localization of peroxisomal proteins in yeast cells.

MiR-4769-3p suppresses adipogenesis in systemic sclerosis by negatively regulating the USP18/VDAC2 pathway.

Systemic sclerosis (SSc) is an autoimmune disease affecting multiple tissues. The underlying causes and mechanisms of subcutaneous adipose tissue (SAT) loss in SSc remain unclear. Recent studies have highlighted the role of microRNAs in adipogenesis. Our study found that miR-4769-3p was upregulated in SSc patients and its silencing promoted SAT recovery in bleomycin-induced SSc mice, suggesting that miR-4769-3p might affect adipogenesis in SSc. Manipulating miR-4769-3p expression in 3T3-L1 cells revealed that its inhibition enhanced adipogenesis, while its overexpression weakened it. Further investigations showed that miR-4769-3p bound to 3'UTR of ubiquitin-specific protease-18 (USP18), inhibiting its expression, while USP18 interacted with voltage-dependent anion channel-2 (VDAC2), both of which were reduced in SSc. Silencing either USP18 or VDAC2 attenuated adipogenesis. Notably, USP18 inhibited VDAC2 ubiquitination and degradation, whereas miR-4769-3p reversed the VDAC2-induced elevation of adipogenesis, suggesting that miR-4769-3p inhibited adipogenesis by negatively regulating the USP18/VDAC2 pathway, providing a potential therapeutic target for SSc.

Virus-encoded glycosyltransferases hypermodify DNA with diverse glycans.

Enzymatic modification of DNA nucleobases can coordinate gene expression, nuclease protection, or mutagenesis. We recently discovered a clade of phage-specific cytosine methyltransferase (MT) and 5-methylpyrimidine dioxygenase (5mYOX) enzymes that produce 5-hydroxymethylcytosine (5hmC) as a precursor for enzymatic hypermodifications on viral genomes. Here, we identify phage MT- and 5mYOX-associated glycosyltransferases (GTs) that catalyze linkage of diverse sugars to 5hmC nucleobase substrates. Metavirome mining revealed thousands of biosynthetic gene clusters containing enzymes with predicted roles in cytosine sugar hypermodification. We developed a platform for high-throughput screening of GT-containing pathways, relying on the Escherichia coli metabolome as a substrate pool. We successfully reconstituted several pathways and isolated diverse sugar modifications appended to cytosine, including mono-, di-, or tri-saccharides comprised of hexoses, N-acetylhexosamines, or heptose. These findings expand our knowledge of hypermodifications on nucleic acids and the origins of corresponding sugar-installing enzymes.

Management of ultrarare inherited arrhythmia syndromes.

Ultrarare inherited arrhythmia syndromes are increasingly diagnosed due to increased awareness as well as increased availability and reduced cost of genetic testing. Yet by definition, their rarity and heterogeneous expression makes development of evidence-based management strategies more challenging, typically employing strategies garnered from similar genetic cardiac disorders. For the most part, reliance on anecdotal experiences, expert opinion, and small retrospective cohort studies is the only means to diagnose and treat these patients. Here we review the management of specific ultrarare inherited arrhythmic syndromes together with the genetic and molecular basis, which will become increasingly important with the development of targeted therapies to the correct the biological basis of these disorders.

Geographical isolation and hyperendemicity of Hepatozoon felis: Epidemiological scenario in Skopelos, Greece, and phylogenetic analysis.

Feline hepatozoonosis is a vector-borne disease caused by different species of the genus Hepatozoon, i.e. Hepatozoon felis, Hepatozoon silvestris and Hepatozoon canis. Knowledge on the biology, epidemiology and taxonomy of Hepatozoon spp. is still limited, despite the fact that the number of documented Hepatozoon spp. infections in domestic cats increased in recent years in different countries. This study was carried out to evaluate the prevalence and the genetic profile of Hepatozoon spp. in cats living on the island of Skopelos, Greece. Individual blood samples were collected from 54 owned cats and were subjected to Giemsa-stained blood smear examination to investigate the presence of Hepatozoon spp. gamonts and to a specific PCR protocol targeting the 18S rRNA gene of Hepatozoon. A total of 45 cats (83.3%) were found infected by Hepatozoon spp. by at least one of the methods applied. In particular, 43 (79.6%) of the cats were PCR-positive, and in 6 (11.1%) cats gamonts of Hepatozoon spp. were found in the blood smears. A total of 26 H. felis sequences were obtained and the presence of three undescribed single nucleotide polymorphisms were detected. The present results indicate that H. felis species complex may be hyperendemic in isolated/confined areas. In such contexts, geographical isolation may favor the origin of new genotypes or haplotypes or even new species.

Protocol for in vivo nucleic acid delivery utilizing the rolling microneedle electrode array.

Electroporation temporarily enhances cell membrane permeability and promotes the absorption of external molecules. We have developed a device termed the rolling microneedle electrode array (RoMEA) that combines a densely arranged microneedle array of electrodes with rolling structures. Use RoMEA to create uniform skin micropores for efficient, low-damage transfection of nucleic acids over extended areas of the body. We describe in detail the design, fabrication, and assembly of the device and the application of in vivo electroporation of nucleic acids. For complete details on the use and execution of this protocol, please refer to Tongren Yang et al. 1.