Molecular Biology Reports

PurposeGlasdegib is a Sonic Hedgehog (SHH) pathway inhibitor used for treating newly diagnosed acute myeloid leukemia in elders or patients unfit for intensive chemotherapy. This study sought to demonstrate growth inhibition and increased apoptosis of B-cell acute lymphoblastic leukemia (B-ALL) in vitro under glasdegib, alone and combined with inotuzumab, using a novel co-culture system and validated chemosensitivity testing model to determine whether glasdegib with and without inotuzumab may represent a promising treatment strategy in B-ALL. MethodsSeven blood and marrow samples from B-ALL patients were co-cultured with HS-5 stromal cells in a co-culturing system designed to mimic the tumor microenvironment to maintain B-ALL cell viability for chemosensitivity testing under glasdegib and inotuzumab. ResultsCo-culturing improved B-ALL viability from four to nine days. Dosage-dependent responses to glasdegib were consistent among B-ALL samples on day four based on culture viability, and varied based on expressions of SSH genes GLI1, GLI3, SMO, and PTCH1. Combination with inotuzumab had varied effects on treatment response. ConclusionCo-culturing B-ALL cells with HS-5 stromal cells improves B-ALL growth and viability. Glasdegib with and without inotuzumab treatments impact the viability of co-cultured B-ALL cells by day four. SHH gene expressions suggest different B-ALL patients may be sensitive or resistant to glasdegib and inotuzumab.

HCT116 is a colorectal cancer cell line frequently used in anti-tumor drug development experiments as well as in studies of the molecular machinery of eukaryotic cells. It is well characterized by the presence of several single-nucleotide and short mutations in multiple oncogenes and tumor suppressor genes, including KRAS, PIK3CA, MLH1, CTNNB1, CDKN2A, TGFBR2, and BRCA2. However, its landscape of large genomic rearrangements (LGRs) and copy number variants (CNVs) is still far from being fully understood. Therefore, the aim of this study was to identify LGRs and CNVs in several HCT116 cell line samples using Oxford Nanopore sequencing technology, including three samples from the SRA NCBI database, and to compare common and unique variants across all samples. Using the recently developed eLaRodON tool, we identified 22,666 common LGRs, among which more than 70% of tandem duplications and deletions larger than 80 kb were confirmed by CNV analysis. Among LGRs affecting protein-coding sequences, two in-frame rearrangements were identified: a deletion of exons 4-6 and a duplication of exon 10 in the CCSER1 gene, which encodes a cell division regulator protein. Given its high rearrangement rate in various tumors and the clinical significance of its overexpression, this finding may be potentially useful in future research on this cell line. Regarding differences between samples, we found that LGRs in the laboratory sample and in one of the three SRA NCBI samples occurred more frequently via ALR/Alpha repeats than via Alu repeats, in contrast to common LGRs and those unique to the other samples, a finding that may indicate the presence of unique mechanisms of genomic instability. Thus, this study reveals a broad spectrum of large genomic rearrangements and copy number variants that can be identified in the HCT116 cell line using Oxford Nanopore sequencing, including rearrangements specific to distinct cell line samples.

Parathyroid hormone-related peptide (PTHrP), encoded by PTHLH, has been implicated in tumor progression through its involvement in epithelial-mesenchymal transition (EMT), angiogenesis, and tumor cell migration. Previous experimental studies suggest that PTHrP may promote these processes in colorectal cancer (CRC), partly through the modulation of factors such as secreted protein acidic and rich in cysteine (SPARC) and vascular endothelial growth factor (VEGFA). These events play a key role in the acquisition of an aggressive phenotype in our experimental models. In this study, we performed an integrative in silico analysis of multiple transcriptomic datasets to investigate the potential role of PTHLH in CRC. Differential expression analysis identified a set of consistently dysregulated genes across independent datasets. Functional enrichment and network analyses revealed that PTHLH expression is associated with biological processes related to extracellular matrix remodeling, EMT, and angiogenesis. Correlation analyses showed a positive association between PTHLH and SPARC expression, while network-based approaches suggested a potential functional connection with VEGFA. To assess the clinical relevance of these findings, survival analysis was performed using publicly available datasets. High expression levels of PTHLH, SPARC, and VEGFA were significantly associated with reduced overall survival in patients. Notably, a combined gene signature based on these three factors demonstrated a stronger prognostic effect than individual genes, indicating enhanced predictive value. These findings suggest that PTHrP is associated with molecular pathways involved in tumor progression and, together with SPARC and VEGF, may contribute to a coordinated regulatory axis with prognostic relevance in CRC, warranting further experimental validation.

The current "gold standard" for diagnosing and assessing treatment response is tumor biopsy; however, biopsies are not always feasible, safe or easily repeated during treatment. Utilization of peripheral blood mononuclear cells (PBMCs) as a surrogate for tumor biopsy allows for longitudinal sampling and is a safer, more readily available option. However, collection conditions, sample transfer time across multiple clinical sites, and PBMC processing conditions are external pre-analytical factors that must be understood and controlled to mitigate bias in downstream functional analyses. This study aims to systematically evaluate the pre-analytical variables affecting PBMC integrity and functional immune readouts as a prerequisite for downstream translational biomarker applications. Peripheral blood samples were collected from 80 treatment-naive patients with a diagnosis of head and neck squamous cell carcinoma. Blood was collected in cell preparation tubes (BD Vacutainer(R) CPT), potassium ethylenediaminetetraacetic acid (EDTA), or sodium heparin (SH) tubes and diluted 1:1 with sterile PBS or remained undiluted. PBMCs were processed and cryopreserved immediately or held for 8- and 24-hours before processing. PBMC viability was measured at cryopreservation and upon thawing. CD8+ T cells or natural killer (NK) cells derived from PBMCs were subjected to cytotoxicity assays using flow cytometry. CPT tubes provided lower cell viability and yield at cryopreservation and upon thaw compared to EDTA and SH tubes while dilution had no effect on viability. NK cell cytotoxicity was similar between EDTA and SH tubes irrespective of dilution. However, diluted EDTA tubes resulted in lower T cell cytotoxicity after 24-hour hold. Viability and NK and T cell cytotoxicity were equivalent between cryopreserved PBMCs that were processed immediately or processed after 8- or 24-hour hold. Here we report cryopreservation methods for reproducibility of viable cells that maintain functional immunological capacity even after significant delay in processing allowing flexibility and feasibility for collection from multiple clinical sites for deferred processing.

BackgroundIn acute myeloid leukemia studies, event-free survival (EFS) is defined as time until treatment failure, relapse, or death, whichever occurs first. Since 2020 and 2022, respectively, the US Food and Drug Administration and the European LeukemiaNet recommend analysing treatment failures as day-1 events. This data modification can lead to a potentially large drop in the estimated EFS at day 1. If censoring occurs, the Kaplan-Meier estimator obtained from the recoded data underestimates this drop. Our aim is to obtain an unbiased estimate for EFS as basis for further inference. MethodsWe define "event on day 1" as one event type and " event after day 1" as a competing event in the original data and use the Aalen-Johansen estimator of the cumulative incidence curve to estimate event-specific transition probabilities, which are combined in one EFS estimate. To analyse effects on day 1 treatment failure and other post-day-1 EFS events separately, a formal link to cure models is established by equating treatment failures with the "cured" proportion in cure model terminology. Additionally, a variance estimator, confidence intervals, confidence bands, and simultaneous testing procedures are derived. ResultsOur new estimation method differs from the Kaplan-Meier estimator in settings in which some treatment failures are censored, as in the interim analysis of the AMLSG 09-09 study. If almost no treatment failures are censored, the two estimation methods do not differ. The cure model and simultaneous testing are able to estimate effects on day 1 treatment failure and other post-day-1 EFS events separately and function independently of whether data is modified. ConclusionsThe Kaplan-Meier estimator evaluated on the recoded data underestimates the drop at day 1 if treatment failures are censored. With sufficient follow-up, this bias disappears, and results coincide with our novel approach.

Heavy metal ATPases (HMAs) are important group of transmembrane proteins involved in homeostasis of metal ions in plant systems. In this study, a comprehensive analysis of genome assembly (VC1973A v7.1) resulted in the identification of nine HMA genes (VrHMA) and their corresponding proteins in Mungbean, an agronomically important legume crop known for its nutritional values. VrHMA proteins were also characterized based on their biomolecular features, conserved domains and motifs arrangement, transmembrane helices, pore-line helices, subcellular location and occurrence of signal peptides. Based on sequence homology, nine VrHMAs were clustered into two major substrate-specific groups: VrHMA1, VrHMA5 and VrHMA7 were categorized under the Zn/Co/Cd/Pb ATPase group, whereas the remaining six VrHMAs belong to the Cu/Ag subgroup. Gene structure analysis and promoter scanning revealed the structural divergence and presence of various stress-responsive cis-acting elements, respectively. The expression analysis of VrHMA genes in root and leaf tissues, in response to heavy metal (Zn, Cd and Cu) stress, indicates their role in the uptake, transport and sequestration of metal ions. Interestingly, VrHMA5 showed incremental upregulation in roots in response to all three heavy metal stresses, whereas its expression was only upregulated in the leaf tissues under Zn stress, which indicates its role in vascular transport in V. radiata. In addition, this study provides valuable insights into the functional roles of VrHMA genes and will lay a foundation for future genetic improvement in mung bean aimed at enhanced heavy metal stress tolerance and micronutrient homeostasis.

BackgroundRNA editing is a post-transcriptional modification that alters the sequence of an RNA transcript. Two types of RNA editing were found in mammals, involving the enzymatic deamination of either adenosine to inosine (A-to-I) or cytidine to uridine (C-to-U) nucleotides in RNA. A-to-I, which is the most common form of RNA editing, is mediated by the ADAR (adenosine deaminases acting on RNA) family of enzymes, ADAR1, ADAR2, and ADAR3. The editing event alters the hydrogen bond pairing of nucleobases, and the editing site will be recorded as guanosine rather than the original adenosine. Indeed, RNA editing deregulation has been linked to several nervous and neurodegenerative diseases. In this project work is done on Alzheimers disease (AD) and the samples are from anterior cingulate cortex of human brain tissue. AD is the main dementia in the world and a neurodegenerative condition prevalent in the elderly. MethodologyA total of 20 raw RNA-sequencing data samples containing 10 controls and 10 Alzheimers disease (AD) cases were collected from NCBI using SRA Toolkit. Quality assessment was performed using FastQC and processed using Trimmomatic. Alignment was done using STAR RNA-seq aligner. RNA editing detection was performed using REDItools, detected sites were subsequently annotated against the REDIportal database. The resulting control-specific and disease-specific novel editing sites were merged into a single dataset containing exclusively novel, group-specific A-to-I editing events. This merged dataset was subsequently used for downstream feature extraction and machine learning analysis. Probability-based filtering was done to extract high-confidence disease associated sites and their gene list was used for computational level biological validation, pathway and functional enrichment analysis as well as overlap with known AD loci. ResultsRandom Forest showed the highest accuracy score (0.804) and ROC-AUC score (0.854). Most important features that differentiated control and diseased novel sites in random forest were coverage ([~]0.35), editing level ([~]0.33) and GC content ([~]0.15). The AEI mean values is higher in both male and female diseased cases ([~]0.48-0.50) but less in male and female control cases ([~]0.14-0.21). The mean values of ADAR1_CPM higher in control cases (123.65-143.30) and is less in diseased cases (88.35-97.93), ADAR2_CPM is almost equal in all cases ([~]3.7-4.7) and ADAR3_CPM is very less in all the cases ([~]0-0.02). Most candidate editing site were present in exon ([~]62-67 %) CDS regions ([~]17-21%) and relatively smaller fraction of gene ([~]15-16 %). Editing alterations preferentially affect molecular systems governing synaptic structure, neurotransmission, and central nervous system integrity. In the main set -of the 2576 high-confidence genes identified, 33 overlapped with AD GWAS loci. In the core set -of the 1367 high-confidence genes identified, 11 overlapped with AD GWAS loci. ConclusionFeature like coverage, editing level and GC content contributed most. Alu sites are negligible as compared to non-alu sites but the AEI mean values are higher in diseased cases than in control cases. The mean values of ADAR1_CPM are higher than ADAR2_CPM and ADAR3_CPM.Sex does not play a major factor. High-confidence disease-associated RNA editing sites are strongly biased toward transcript-centric regions, particularly exons, with a notable subset affecting coding sequences. Importantly, enrichment of neurodegeneration-associated pathways and cognition-related human phenotypes further supports the disease relevance of these gene networks. RNA editing events in Alzheimers cortex may represent a regulatory mechanism largely independent of inherited genetic susceptibility loci.

BackgroundConventional models of human ribosomal DNA (rDNA) array organization have historically depended on transcription-centric boundaries, partitioning the unit into a [~]13 kb rDNA transcription region and a monolithic [~]31 kb intergenic spacer (IGS). While our previous identification of Duplication Segment Units (DSUs) mapped these arrays based on an intuitive analysis of the microsatellite density landscape of the complete reference human genome, our present deep mining of this landscape has revealed a more accurate rDNA Gene Unit Pattern. Methods & ResultsIn this study, we conducted a deep mining analysis of our previously established microsatellite density landscape of the T2T-CHM13 assembly, focusing specifically on nucleolar organizing regions (NORs). We suggest a more accurate rDNA Gene Unit Pattern containing a (CTTT)n microsatellite aggregation ahead of the rDNA gene and a (CT)n microsatellite aggregation behind the gene, rather than a pattern featuring an IGS region inserted between two rDNA genes. ConclusionsA correct rDNA gene pattern of the human genome probably includes a (CTTT)n microsatellite aggregation ahead of the gene and a (CT)n microsatellite aggregation behind it, which possibly constitute cis- and trans-regulating regions; the (CTTT)n and (CT)n microsatellite aggregations may provide two different local stable DNA structures for regulatory protein binding.

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

Some Anopheles species that act as malaria vectors are members of species complexes, a concept whereby sibling species cannot be differentiated solely on the basis of morphological characters. Therefore, species complexes represent a major problem in malaria vector control, because within an Anopheles complex, vectors cannot be differentiated from non-vector species, unless molecular techniques are used to identify them. The Anopheles tessellatus species complex is an important potential vector in South, East, and Southeast Asia, including certain regions of Indonesia. However, no in-depth studies have been conducted on this species complex in that country. Therefore, this study investigated the taxonomic status of An. tessellatus from diverse populations across five Indonesian islands (Sumatra, Java, West Nusa Tenggara, East Nusa Tenggara, and Sulawesi) and identified interpopulation genetic variation based on molecular data of the ITS2, COI, and COII genes. Phylogenetic relationships were constructed using the Maximum Likelihood method. Haplotype and network analysis were also conducted. The results indicate that An. tessellatus constitutes a monophyletic group comprising three well-defined lineages that exhibit clear intraspecific genetic differentiation. Cluster 1 corresponds to the population of Sumatra, Cluster 2 represents population from Sulawesi, and Cluster 3 encompasses populations from Java, West Nusa Tenggara, and East Nusa Tenggara. These findings demonstrate high haplotype diversity and low nucleotide diversity within the species. Populations from West Sumatra, Manado, Tojo Una - Una, and North Morowali (Sulawesi) have the potential for speciation with a genetic distance of 0.61 - 0.94% for COI, between 0.81 - 0.95% for ITS2, and between 0.62 - 0.71% for COII. These findings underscore the need for further integrative studies to obtain a more comprehensive understanding of the An. tessellatus complex in Indonesia and its role in malaria transmission.

BackgroundAnthracyclines are central to childhood cancer therapy but predispose patients to cardiotoxicity leading to long-term cardiovascular risk. Endothelial injury and impaired repair contribute to this, yet pediatric data remain limited. ObjectiveTo longitudinally assess endothelial injury and repair in childhood cancer patients treated with anthracyclines by quantifying circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs). MethodsIn a single-centre retrospective cohort, children (<18 years) diagnosed with leukemia (n=35) or lymphoma (n=13) were studied at four timepoints: pre-treatment ("Pre"), [~]1-month- ("End"), 3 months- (3M), and 1 year- (1Y) post-treatment. Peripheral blood mononuclear cells were analyzed by flow cytometry to quantify CECs and EPCs, and EPC fate was assessed by p16 (senescence) and Annexin V (apoptosis). Cardiac injury biomarkers and left ventricular function were assessed at each timepoint. ResultsLongitudinal trends of CEC and EPC counts were similar between leukemia and lymphoma participants. CECs were highest at pre-treatment and declined significantly thereafter, though they remained marginally elevated during remission compared with healthy controls, indicating that endothelial damage had largely subsided following treatment. EPCs were also highest at pre-treatment and decreased to levels below healthy controls during remission, suggesting impaired baseline endothelial maintenance and repair. Furthermore, EPCs were predominantly senescent up to 1-year post-treatment. ConclusionsEndothelial injury resolves by treatment completion, but repair remains impaired during remission with EPC pools dominated by senescent cells. This suggests defective endothelial regeneration, rather than persistent injury, drives long-term cardiovascular complications and underscores the need to restore EPC viability and function in childhood cancer survivors.

The molecular basis of triple-negative breast cancer (TNBC), a highly aggressive and therapy-resistant subtype of breast cancer, is poorly understood. This study aims to identify key genes and pathways involved in TNBC development and progression using a systems biology approach followed by experimental validation. Here, two transcriptome microarray datasets from the GEO database were analysed using the R package LIMMA to detect differentially expressed genes (DEGs) in TNBC tumors. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses using the DAVID database were performed to identify DEGs regulated biological functions and pathways. Further, a protein-protein interaction (PPI) network was constructed using the STRING online database, and the topological properties were determined using MCODE and Cytohubba plug-ins. The expression and the prognostic value of the hub genes were validated using the Cancer Genome Atlas (TCGA) survival analysis. We found 727 DEGs, of which 473 were downregulated and 254 were upregulated in TNBC vs. non-TNBC samples. The GO and KEGG analyses indicated that the DEGs were mainly related to cell adhesion, tumorigenesis, and cellular immunity. The PPI network had shown six hub genes, namely CCND1, CDH1, ESR1, FN1, IL6, and PPARG, as the top key regulators. All these genes were validated by quantitative real-time PCR in the TNBC cell line using non-TNBC cell line as a calibrator, and the obtained results were in accordance with the bioinformatics data. This information may contribute to understanding the various molecular mechanisms that drive the development and progression of TNBC tumors.

Biological sequences are known to be not random. Thus, the comparison of in silico restriction fragment distributions of random and biological sequences may be an indicator of this non-randomness. Our analyses show that for most of the tested combinations of restriction enzyme and genome sequence the fragments per Megabase of the biological sequence deviate at least more then 10% from the corresponding random sequence. This deviation goes into both directions, i.e. clearly increased values are as common as clearly decreased values. Although there is no species- or restriction-enzyme-specific effect, a clear impact of the GC content both of the restriction site and of the genome sequence can be seen. In contrast to the random sequences, the genome sequences show distinct peaks in their fragment length distributions, hinting to repetitive elements such as transposons.

Pulmonary vascular remodelling is common in patients with chronic obstructive pulmonary disease (COPD). Vascular endothelial growth factors (VEGFs) are key mediators in angiogenesis and vascular remodelling and exist in different isoforms. VEGF-A is the most potent angiogenic member binding to VEGF receptor 2 (VEGFR2). There are, however, few studies on other isoforms, as VEGF-C, and its receptor VEGFR3 in COPD and subsequent impact of cAMP therapies on VEGF isoforms. Our aim was to evaluate the VEGF isoform synthesis in primary distal lung fibroblasts from control subjects (non-smokers (n=6) and ex-smokers (n=4), and COPD subjects with GOLD stage II (n=4) or GOLD stage IV (n=6), and the expression of VEGFR2 and VEGFR3 in human lung tissue. Primary lung fibroblasts were exposed to the cAMP generating therapies formoterol, iloprost, or roflumilast, the adenylyl cyclase activator forskolin or to transforming growth factor (TGF)-b1. VEGF isoforms were evaluated with ELISA. VEGF-C release was not significantly altered by TGF-{beta}1, in contrast to the increased levels of VEGF-A, in all fibroblasts. VEGF-C was significantly decreased by iloprost, forskolin and formoterol, whereas VEGF-A was significantly increased by iloprost and forskolin, with differences in release pattern between and within fibroblasts from control and COPD subjects. Exposure to VEGF-C specifically towards VEGFR3 decreased proliferative rate in human lung fibroblasts and bronchial epithelial cells. VEGFR2 and VEGFR3 were both present in parenchymal lung tissue and VEGFR2 in pulmonary blood vessels. in both healthy and COPD, whereas there was elevated expression of VEGFR3 in bronchial epithelium. In conclusion, TGF-{beta}1 and cAMP generating compounds have significant effects on VEGF-C and VEGF-A synthesis, which appear dysregulated in lung fibroblasts from ex-smokers and patients with COPD. Increased VEGFR3 expression in the bronchial epithelium in lung tissue, and studies into their functional impact, warrants further investigations.

While induced pluripotent stem cells (iPSCs) have gained popularity in studying neurodegenerative diseases, the heterogeneity of stem cells used across studies impacts cross-study comparison. The iPSC Neurodegenerative Disease Initiative (iNDI) selected the KOLF2.1J cell line and prioritized its use as a reference standard for studying the effects of pathogenic variants on cell biology due to its stability and neutral neurodegenerative disease genetic risk. This cell line, and its derivatives expressing over 100 variants related to Alzheimers disease, Parkinsons disease, and other neurological diseases, are available for academic and industry access. Current genomic data analyses are limited by the use of a human reference genome that does not capture the complete genetic background of a given iPSC line. While in the future this issue may be partially mitigated by the creation of a comprehensive human pangenome, previous work has shown that generating custom genomes is of value both to characterize the variation present and to serve as a more appropriate genomic reference. Here, we generated and characterized a custom complete genome assembly from KOLF2.1J. Mapping of sequencing reads to a personalized diploid assembly results in more comprehensive mapping compared to traditional linear references (i.e GRCh38). In addition, we provide a comprehensive custom gene annotation along with isoform expression and differential methylation analyses across multiple cell types. The assembly and all additional data is browsable and publicly available. This resource will enable more accurate investigation of the KOLF2.1J cell line and any genomics data generated compared to using traditional generalized references, while also serving as a foundational approach for establishing custom reference assemblies for other high-value iPSC lines.

The paper presents the results of a comparative analysis of gene networks activated by water stress and low temperatures in extensive (Saratovskaya 29, S29) and intensive (Yanetskis Probat, YP) wheat varieties during the seedling development stage. It is concluded that the creation of the S29 variety, which occurred through complex stepwise hybridization and selection for morphological traits, productivity, and grain quality traits, resulted in the emergence and inheritance of regulatory gene networks involving proteins with the CC domain, as well as the BTB/POZ and TAZ domains, which have an increased affinity for transcription factors involved in the transcriptomic response to changing external conditions. It was established that, at the transcriptomic level, the S29 variety is characterized by a transition to an energy saving mode to maintain the activity of the Calvin-Benson cycle under the water deficit conditions and the inhibition of proteolytic processes at low temperatures. The transcriptional response of the high-yielding YP variety to 24-hour low-temperature treatment was more active and involved a larger number of genes compared to the S29 variety. Identifying varietal variability in molecular genetic mechanisms of resistance to abiotic stressors facilitates the development of marker-assisted and genomic selection technologies for common wheat. Key messageThe extensive S29 variety was characterized by its transition to energy-saving mode to maintain the Calvin-Benson cycle under water deficit and a reduction in proteolytic processes under low temperature.

Garcinia binucao (Blanco) Choisy is an indigenous species endemic to the Philippines. Its fruit is traditionally used as a souring agent in local cuisine and has been reported to possess nutritional and medicinal properties. Despite its ethnobotanical significance and promising bioactive properties, the species remains underutilized. To date, no genomic resources have been published for G. binucao, limiting its application in food systems, genetic studies, and conservation programs. This study reports the first complete chloroplast genome of G. binucao from an accession conserved at the Institute of Plant Breeding, University of the Philippines Los Banos. The assembled plastome is circular with a length of 156,570 base pairs (bp). It displays the typical quadripartite structure of most angiosperms, consisting of a large single-copy (LSC) region (85,357 bp), a small single-copy (SSC) region (17,129 bp), and a pair of inverted repeats (IR), each 27,042 bp in size. A total of 128 genes were annotated, including 83 protein-coding genes, 37 transfer RNAs (tRNAs), and eight ribosomal RNAs (rRNAs), consistent with the majority of Garcinia species. Of the protein-coding genes, 45 are involved in photosynthesis, 28 genes for self-replication, five genes with conserved open reading frames, and five genes are associated with other functions. The GC content was 36.2%. Leucine (10.6%) was the most abundant amino acid, with a codon usage bias toward UUA. Additionally, 98 simple sequence repeats (SSRs) were detected, 88.78% consisting of A/T motifs. Phylogenomic analysis based on assembled plastome and publicly available cpDNA sequences of 17 other species in the order Malpighiales revealed that G. indica is the closest relative of G. binucao. These findings provide a framework for future research on the species, including its conservation and potential use as a genetic resource.

At the Wisconsin National Primate Research Center, we have identified a family of rhesus carrying the microtubule-associated protein tau (MAPT) R406W mutation linked to frontotemporal dementia (FTD). Rhesus induced pluripotent stem cells (RhiPSCs) derived from these monkeys present a unique opportunity for in vitro modeling and comparison with cells derived from MAPT R406W human carriers. Here, we report the development of a reproducible method to generate RhiPSCs compliant with the standards of the International Society for Stem Cell Research (ISSCR) to support in vitro modeling of FTD-MAPT R406W. Our stepwise approach identified efficient methods for fibroblast derivation, fibroblast reprogramming to RhiPSC, and RhiPSC maintenance over continued culture. To derive fibroblasts from MAPT wild type (WT) and R406W monkeys, a combination of manual processing and overnight enzymatic digestion was required to maximize the number of low passage fibroblasts available for reprogramming. Fibroblast reprogramming to RhiPSC using Sendai viral vectors versus oriP/EBNA1 episomal plasmids revealed the latter as most efficient. Electroporation conditions for oriP/EBNA1 reprogramming were optimized to maximize plasmid uptake and cell survival. Ultimately, eight RhiPSC lines were derived from 4 donor rhesus monkeys (n=2 WT, n=2 R406W; two clonal lines per donor) and fully characterized according to ISSCR standards. RhiPSC stemness and genetic stability was best maintained on mouse embryonic fibroblast feeders in Universal Primate Pluripotency Stem Cell medium, as opposed to Essential 12 medium supplemented with IWR1, which produced cytogenetic abnormalities. Rhesus neural progenitor cells were generated using a monolayer protocol and expressed PAX6 and NESTIN after 21 days of differentiation. Our reliable method will be useful to labs seeking to derive RhiPSCs for preclinical studies. Overall, the RhiPSCs generated from MAPT R406W carriers will be a critical resource for evaluating the molecular underpinnings of tau-related neurodegeneration across primate species.

Obtaining tomato plants with firm and intact fruit is one of the main goals in tomato breeding programs. Achieving these goals through conventional breeding is time-consuming and can lead to the loss of unwanted traits. In other hand, consumers are concerned about the presence of transgenic elements in plants acquired through RNA interference. The use of CRISPR/Cas9 technology has made it possible to overcome the above-mentioned shortcomings. In this study, the {beta}-D-N-acetylhexosaminidase ({beta}-hex) gene, which is involved in tomato fruit ripening, was knocked out using CRISPR/Cas9. In the resulting mutant plant genome, an indel mutation was found in exons 1 and 2 of the {beta}-hex gene. Plants with a mutation in their genome were observed to have increased fruit firmness and shelf life compared to control plants without affecting fruit quality.

Dengue virus (DENV) infection generates a significant health burden throughout the world, and there are no clinically approved antiviral drugs, as of now. The virus also depends on lipid metabolism in the host to conduct effective replication and this makes lipid-directed compounds promising as therapeutic options. We assessed the antiviral effect of lauric acid, a 12-carbon medium-chain fatty acid, against DENV serotype 2 (DV2) in the presence of a stable cell line, DV2-replicon, expressing all the non-structural proteins (NS1-NS5) and a luciferase reporter. Active viral replication in replicon cells was established by morphological examination and immunofluorescence of cells. The MTT assay was used to determine the cytotoxicity of lauric acid revealing the LD50 of 2.52 uM, so higher concentrations were toxic as the effect of the drug is dose-related. The antiviral effect was tested through replicon inhibition (luciferase) assay which showed an incredible inhibition of viral RNA replication with a IC50 of 1.70 uM and this is equivalent to antiviral mycophenolphycic acid. The cytopathic effects, as well as a decrease in the activity of luciferase, proved the presence of viral translation and replication inhibition within the process of the treatment of the lauric acid. These results propose that lauric acid has cytotoxic and antiviral dual effect and can be a possible inhibitor of DENV replication. The toxicity needs to be reduced and future research is necessary to explain its molecular pathway and also to come up with the best delivery methods. IMPORTANCEDengue virus (DENV) remains a significant health challenge to the world since there are no effective antiviral agents. This work will recognize lauric acid as a possible dengue virus replication inhibitor in a model of a DV2 replicon, exhibiting antiviral action that is similar to that of mycophenolic acid. These results support lipid-directed compounds as potential dengue antiviral targets, but more research is needed to minimize toxicity and better understand the molecular mechanism of action.