Mutations In Sequence Research Articles

Probing protein stability: towards a computational atomistic, reliable, affordable, and improvable model.

We present an improved application of a recently proposed computational method designed to evaluate the change of free energy as a function of the average value of a suitably chosen collective variable in proteins. The method is based on a full atomistic description of the protein and its environment. The goal is to understand how the protein melting temperature changes upon single-point mutations, because the sign of the temperature variation will allow us to discriminate stabilizing vs. destabilizing mutations in protein sequences. In this refined application the method is based on altruistic well-tempered metadynamics, a variant of multiple-walkers metadynamics. The resulting metastatistics is then modulated by the maximal constrained entropy principle. The latter turns out to be especially helpful in free-energy calculations as it is able to alleviate the severe limitations of metadynamics in properly sampling folded and unfolded configurations. In this work we apply the computational strategy outlined above in the case of the bovine pancreatic trypsin inhibitor, a well-studied small protein, which is a reference for computer simulations since decades. We compute the variation of the melting temperature characterizing the folding-unfolding process between the wild-type protein and two of its single-point mutations that are seen to have opposite effect on the free energy changes. The same approach is used for free energy difference calculations between a truncated form of frataxin and a set of five of its variants. Simulation data are compared to in vitro experiments. In all cases the sign of the change of melting temperature is reproduced, under the further approximation of using an empirical effective mean-field to average out protein-solvent interactions.

The clinical value of tumor-informed minimal residual disease detection in sarcoma.

11510 Background: The high recurrence and metastasis rates lead to poor survival of patients with sarcoma. At present, circulating tumor DNA-based minimal residual disease (MRD) has been studied in varies solid tumors and proved to be valuable in guiding treatment and predicting recurrence, but its application in sarcomas is rarely reported. Methods: Tumor tissue samples were used for whole exome sequencing (WES) and clonal mutations were selected for the customized MRD panel. Blood samples were collected for MRD detection. Experiments were conducted at OrigiMed, a College of American Pathologists accredited and Clinical Laboratory Improvement Amendments certified laboratory. Results: A total of 84 sarcoma patients including 47 bone tumor (BT), 32 soft-tissue sarcoma (STS), and 5 undifferentiated small round cell sarcoma (USRCS) patients were enrolled. Patients consisted of 49 (58.3%) males and 35 (41.7%) females, with a median age of 23 years old (1-85 years old). Based on WES, we found that 64% (4936/7715) of the mutated genes were unique to each patient, and 70.4% (859/1220) of selected tumor-informed single nucleotide variants (SNV) were variants with unknown significance, suggesting that tumor-informed MRD is superior to panel-based MRD in sarcomas. In this cohort, 78.6% (66/84) of patients successfully constructed MRD panels, including 85.1% (40/47) of BT patients, 75% (24/32) of STS patients, and 40% (2/5) of USRCS patients. The main reason for failure was the scarcity of SNVs detected. Preoperative MRD detection was performed on 6 cases and revealed a positive rate of 100%. MRD detection was performed twice on 2 patients during preoperative neoadjuvant therapy. For the patient with constant allele frequencies detected throughout the treatment course, imaging showed tumor enlargement; While for another patient with a trend of reduction in allelic frequency, imaging showed tumor shrinkage and necrotic tumor tissue was observed in surgery one month later. These two patients had not recurred after surgery at the time of analysis. Post-surgery MRD detection were performed on 27 patients, of which 1 STS and 7 BTs were positive (30%, 8/27). Three of them confirmed recurrence by imaging at 12, 30, and 101 days after detection of positive MRD, respectively. However, there were still no imaging recurrence in the other patients so far, including the 5 remaining patients with positive MRD and 19 patients with negative MRD. Follow-up is still ongoing. Conclusions: MRD tests had been successfully performed in most of the sarcoma patients. Due to the heterogeneity of sarcomas, about 64% of mutated genes of sarcomas are unique individually, which may be more suitable for monitoring with tumor-informed MRD panel. Tumor-informed MRD could be used to predict the efficacy of preoperative neoadjuvant therapy and discover recurrence earlier than imaging in sarcoma.

Association of weight loss with risk of myelodysplastic syndromes (MDS) in adults with obesity: Insights from the SPLENDID cohort.

e18865 Background: MDS arises from pre-existing somatic mutations or clonal hematopoiesis (CH). A normal body mass index (BMI) is strongly associated with a lower prevalence of CH. We hypothesized that intentional weight loss (IWL) might be associated with lower incidence of MDS and studied this in the context of bariatric surgery (BSx) among adults with obesity. Methods: In the SPLENDID (Surgical Procedures and Long-term Effectiveness in Neoplastic Disease Incidence and Death) matched cohort study, adults with BMI ≥35Kg/m2 who had BSx (ages 18-80) during 2004-2017 were included. Each patient (pt) who underwent BSx was matched with propensity score by the nearest-neighbor method to five pts who did not undergo BSx for their obesity, resulting in a total of 30318 pts. MDS diagnosis was made using contemporary WHO criteria and myeloid sequencing mutation data were analyzed. Multivariable cox regression analysis estimated time to MDS as the primary endpoint. Results: 5053 adults with obesity had BSx while 25265 adults underwent nonsurgical (NS) care. Median age and BMI were 46 years (y) and 45kg/m2. The majority were females (77%); and had white (73%) race. Follow-up was over 17y period with median (IQR) of 6.1 (3.8-8.9)y for the entire cohort. BSx-group had 19.2% greater WL at 10-y [mean between-group difference: 24.8 (95% CI, 24.6-25.1) kg]. One patient (male) in the BSx-group vs twenty-four patients (twelve females) in the NS-group developed MDS (incidence rate: 0.03 vs 0.14, per 1000 person-years). A fully adjusted hazard ratio was 0.14 (95%CI 0.02-1.14), which did not reach conventional level of statistical significance (p = 0.066) due to small number of events. A third of MDS pts had high-risk disease per IPSS-R prognostic score (7/20 evaluable pts). 10 (of 11 NS MDS pts with available NGS data) pts had ≥1 nonsynonymous mutations and most pts (6/11) harbored multiple [Median (IQR): 2 (1-3)] high-risk mutations despite a younger median age of the cohort. Conclusions: In this large cohort of adults with obesity, we observed a lower incidence of MDS after IWL achieved with BSx. Higher prevalence of high-risk-MDS mutations in a relatively young NS-cohort with obesity warrants further investigation of effects of IWL on clonal evolution, its clinical relevance, and role of screening in halting progression of CH to MDS via early detection and targeted intervention. [Table: see text]

Evaluating Machine Learning Models on Predicting Change in Enzyme Thermostability

Enzymes are efficient catalysts for biological reactions and can potentially be designed to speed up non-biological reactions, such as reactions in industrial processes. However, physically experimenting with new protein designs is time consuming, and an efficient method to predict protein stability is needed. Our research problem is finding the best machine learning model to predict the change in enzyme thermostability after a single point mutation in the amino acid sequence. We trained several machine learning models and found that the XGBoost model had the best performance with an R2 score of 0.593 (R2 score is a metric where higher is better and a perfect model would have a score of 1).

Improved prime editing allows for routine predictable gene editing in Physcomitrium patens.

Efficient and precise gene editing is the gold standard of any reverse genetic study. The recently developed prime editing approach, a modified CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] editing method, has reached the precision goal but its editing rate can be improved. We present an improved methodology that allows for routine prime editing in the model plant Physcomitrium patens, whilst exploring potential new prime editing improvements. Using a standardized protoplast transfection procedure, multiple prime editing guide RNA (pegRNA) structural and prime editor variants were evaluated targeting the APT reporter gene through direct plant selection. Together, enhancements of expression of the prime editor, modifications of the 3' extension of the pegRNA, and the addition of synonymous mutation in the reverse transcriptase template sequence of the pegRNA dramatically improve the editing rate without affecting the quality of the edits. Furthermore, we show that prime editing is amenable to edit a gene of interest through indirect selection, as demonstrated by the generation of a Ppdek10 mutant. Additionally, we determine that a plant retrotransposon reverse transcriptase enables prime editing. Finally, we show for the first time the possibility of performing prime editing with two independently coded peptides.

Mutations in EFEMP1 in Patients with Juvenile Open-Angle Glaucoma

Determine the role of the EFEMP1 gene in the pathogenesis of juvenile open-angle glaucoma (JOAG). Prospective case-control PARTICIPANTS: 72 JOAG and 215 POAG patients and 362 controls from Iowa. DNA was tested for coding sequence mutations in the EFEMP1gene using Sanger or whole exome sequencing. No non-synonymous EFEMP1 mutations were detected in JOAG patients and one non-synonymous EFEMP1variant (c.146A>C, p.Asp49Ala) was detected in both POAG patients (n= 2, 1.5%) and controls (n=4, 1.1%) at similar frequencies (p = 0.67). EFEMP1 mutations are not a common cause of glaucoma in our cohort from Iowa.

No Difference in the Prevalence of HIV-1 gag Cytotoxic T-Lymphocyte-Associated Escape Mutations in Viral Sequences from Early and Late Parts of the HIV-1 Subtype C Pandemic in Botswana.

HIV is known to accumulate escape mutations in the gag gene in response to the immune response from cytotoxic T lymphocytes (CTLs). These mutations can occur within an individual as well as at a population level. The population of Botswana exhibits a high prevalence of HLA*B57 and HLA*B58, which are associated with effective immune control of HIV. In this retrospective cross-sectional investigation, HIV-1 gag gene sequences were analyzed from recently infected participants across two time periods which were 10 years apart: the early time point (ETP) and late time point (LTP). The prevalence of CTL escape mutations was relatively similar between the two time points-ETP (10.6%) and LTP (9.7%). The P17 protein had the most mutations (9.4%) out of the 36 mutations that were identified. Three mutations (A83T, K18R, Y79H) in P17 and T190A in P24 were unique to the ETP sequences at a prevalence of 2.4%, 4.9%, 7.3%, and 5%, respectively. Mutations unique to the LTP sequences were all in the P24 protein, including T190V (3%), E177D (6%), R264K (3%), G248D (1%), and M228L (11%). Mutation K331R was statistically higher in the ETP (10%) compared to the LTP (1%) sequences (p < 0.01), while H219Q was higher in the LTP (21%) compared to the ETP (5%) (p < 0.01). Phylogenetically, the gag sequences clustered dependently on the time points. We observed a slower adaptation of HIV-1C to CTL immune pressure at a population level in Botswana. These insights into the genetic diversity and sequence clustering of HIV-1C can aid in the design of future vaccine strategies.

IDENTYFIKACJA MUTACJI WYBRANYCH GENÓW POWODUJ ˛ACYCH ZWYRODNIENIE SIATKÓWKI U KOTA DOMOWEGO (FELIS CATUS) – ANALIZA IN SILICO

Retinal degenerations are a series of genetically inherited diseases resulting in significant visual impairment and blindness. Among domestic cat breeds, there are degenerations of different courses associated with mutations in CEP290, CRX, AIPL1 and KIF3B genes. The aim of this study was to design diagnostic tests to identify the mutated alleles. The primers for PCR and restriction enzymes for PCR-RFLP were designed to detect mutations in genes. Mutation in the nucleotide sequence encoding AIPL1 protein causes a change in the protein structure, where a monomer is formed instead of a homodimer. Interactions of CEP290, CRX, AIPL1 and KIF3B proteins with other proteins that play a role in the proper functioning of the retina were observed. The occurring interactions between some of these proteins suggest a possible link between diseases caused by mutations of genes encoding these proteins. In other animal species, co-expression of the analyzed genes with other genes affecting retinal functions was noted.

Relationship between the Reduced Expression of Zinc Finger Protein 668 in Bladder Cancer and Its Invasiveness.

The zinc finger protein 668 (ZNF668) gene encodes a Kruppel C2H2-type zinc-finger protein with 16 C2H2-type zinc fingers. The ZNF668 gene functions as a tumor suppressor gene in breast cancer. We histologically analyzed ZNF668 protein expression in bladder cancer and examined mutations of the ZNF668 gene in 68 cases of bladder cancer. In bladder cancer, the ZNF668 protein was expressed in the nuclei of cancer cells. In bladder cancer with submucosal and muscular infiltration, the expression of ZNF668 protein was significantly lower than that without submucosal and muscular infiltration. Eight heterozygous somatic mutations were detected in exon3 in five cases, and five of the mutations resulted in amino acid sequence mutations. Mutations resulting in amino acid sequence alterations also resulted in lower ZNF668 protein expression in bladder cancer cell nuclei, but no significant association with bladder cancer infiltration was detected. Decreased ZNF668 expression in bladder cancer was associated with submucosal and muscle invasion of cancer cells. Somatic mutations resulting in amino acid mutations in ZNF668 were found in 7.3% of the bladder cancer cases.

Structural Optimization and Interaction Study of a DNA Aptamer to L1 Cell Adhesion Molecule.

The L1 cell adhesion molecule (L1CAM) plays important roles in the development and plasticity of the nervous system as well as in tumor formation, progression, and metastasis. New ligands are necessary tools for biomedical research and the detection of L1CAM. Here, DNA aptamer yly12 against L1CAM was optimized to have much stronger binding affinity (10-24 fold) at room temperature and 37 °C via sequence mutation and extension. This interaction study revealed that the optimized aptamers (yly20 and yly21) adopted a hairpin structure containing two loops and two stems. The key nucleotides for aptamer binding mainly located in loop I and its adjacent area. Stem I mainly played the role of stabilizing the binding structure. The yly-series aptamers were demonstrated to bind the Ig6 domain of L1CAM. This study reveals a detailed molecular mechanism for the interaction between yly-series aptamers and L1CAM and provides guidance for drug development and detection probe design against L1CAM.

Sustainable and high-level microbial production of plant hemoglobin in Corynebacterium glutamicum

BackgroundPlant hemoglobin shows great potential as a food additive to circumvent the controversy of using animal materials. Microbial fermentation with engineered microorganisms is considered as a promising strategy for sustainable production of hemoglobin. As an endotoxin-free and GRAS (generally regarded as safe) bacterium, Corynebacterium glutamicum is an attractive host for hemoglobin biosynthesis.ResultsHerein, C. glutamicum was engineered to efficiently produce plant hemoglobin. Hemoglobin genes from different sources including soybean and maize were selected and subjected to codon optimization. Interestingly, some candidates optimized for the codon usage bias of Escherichia coli outperformed those for C. glutamicum regarding the heterologous expression in C. glutamicum. Then, saturated synonymous mutation of the N-terminal coding sequences of hemoglobin genes and fluorescence-based high-throughput screening produced variants with 1.66- to 3.45-fold increase in hemoglobin expression level. To avoid the use of toxic inducers, such as isopropyl-β-d-thiogalactopyranoside, two native inducible expression systems based on food additives propionate and gluconate were developed. Promoter engineering improved the hemoglobin expression level by 2.2- to 12.2-fold. Combination of these strategies and plasmid copy number modification allowed intracellular production of hemoglobin up to approximately 20% of total protein. Transcriptome and proteome analyses of the hemoglobin-producing strain revealed the cellular response to excess hemoglobin accumulation. Several genes were identified as potential targets for further enhancing hemoglobin production.ConclusionsIn this study, production of plant hemoglobin in C. glutamicum was systematically engineered by combining codon optimization, promoter engineering, plasmid copy number modification, and multi-omics-guided novel target discovery. This study offers useful design principles to genetically engineer C. glutamicum for the production of hemoglobin and other recombinant proteins.

Gene mutation landscape of a rare patient with acute megakaryoblastic leukemia after treatment of intracranial germ cell tumor.

It was first reported that germ cell tumor patients suffer from hematologic malignancies 37 years ago. Since then, the number of relevant reports has increased each year, with most cases being mediastinal germ cell tumor. Theories have been proposed to explain this phenomenon, including a shared origin of progenitor cells, the effects of treatment, and independent development. However, up to now, no widely accepted explanation exists. The case with acute megakaryoblastic leukemia and intracranial germ cell tumor has never been reported before and the association is far less known. We used whole exome sequencing and gene mutation analysis to study the relationship between intracranial germ cell tumor and acute megakaryoblastic leukemia of our patient. We report a patient who developed acute megakaryoblastic leukemia after treatment for an intracranial germ cell tumor. Through whole exome sequencing and gene mutation analysis, we identified that both tumors shared the same mutation genes and mutation sites, suggesting they originated from the same progenitor cells and differentiated in the later stage. Our findings provide the first evidence supporting the theory that acute megakaryoblastic leukemia and intracranial germ cell tumor has the same progenitor cells.

Identification of sequence mutations in Phytophthora cactorum genome associated with mefenoxam resistance and development of a molecular assay for the mutant detection in strawberry (F. × ananassa)

Phytophthora crown rot (PhCR) caused by Phytophthora cactorum is one of the most damaging diseases of strawberry worldwide. Mefenoxam is one of the major fungicides currently used to manage PhCR. However, the emergence and spread of resistant isolates have made controlling the pathogen in the field problematic. In the present study, using whole genome sequencing analysis, mutations associated with mefenoxam-resistant isolates were identified in six different genomic regions of P. cactorum. The 95.54% reads from a sensitive isolate pool and 95.65% from a resistant isolate pool were mapped to the reference genome of P. cactorum P414. Four point mutations were in coding regions while the other two were in noncoding regions. The genes harboring mutations were functionally unknown. All mutations present in resistant isolates were confirmed by sanger sequencing of PCR products. For the rapid diagnostic assay, SNP-based high-resolution melting (HRM) markers were developed to differentiate mefenoxam-resistant P. cactorum from sensitive isolates. The HRM markers R3-1F/R3-1R and R2-1F/R2-1R were suitable to differentiate both sensitive and resistant profiles using clean and crude DNA extraction. None of the mutations associated with mefenoxam resistance found in this study were in the RNA polymerase subunit genes, the hypothesized target of this compound in oomycetes. Our findings may contribute to a better understanding of the mechanisms of resistance of mefenoxam in oomycetes since serves as a foundation to validate the candidate genes as well as contribute to the monitoring of P. cactorum populations for the sustainable use of this product.

Roles for mycobacterial DinB2 in frameshift and substitution mutagenesis.

Translesion synthesis by translesion polymerases is a conserved mechanism of DNA damage tolerance. In bacteria, DinB enzymes are the widely distributed promutagenic translesion polymerases. The role of DinBs in mycobacterial mutagenesis was unclear until recent studies revealed a role for mycobacterial DinB1 in substitution and frameshift mutagenesis, overlapping with that of translesion polymerase DnaE2. Mycobacterium smegmatis encodes two additional DinBs (DinB2 and DinB3) and Mycobacterium tuberculosis encodes DinB2, but the roles of these polymerases in mycobacterial damage tolerance and mutagenesis is unknown. The biochemical properties of DinB2, including facile utilization of ribonucleotides and 8-oxo-guanine, suggest that DinB2 could be a promutagenic polymerase. Here, we examine the effects of DinB2 and DinB3 overexpression in mycobacterial cells. We demonstrate that DinB2 can drive diverse substitution mutations conferring antibiotic resistance. DinB2 induces frameshift mutations in homopolymeric sequences, both in vitro and in vivo. DinB2 switches from less to more mutagenic in the presence of manganese in vitro. This study indicates that DinB2 may contribute to mycobacterial mutagenesis and antibiotic resistance acquisition in combination with DinB1 and DnaE2.

Benchmarking Molecular Dynamics Force Fields for All-Atom Simulations of Biological Condensates.

Proteins containing intrinsically disordered regions are integral parts of the cellular signaling pathways and common components of biological condensates. Point mutations in the protein sequence, genetic at birth or acquired through aging, can alter the properties of the condensates, marking the onset of neurodegenerative diseases such as ALS and dementia. While the all-atom molecular dynamics method can, in principle, elucidate the conformational changes that arise from point mutations, the applications of this method to protein condensate systems is conditioned upon the availability of molecular force fields that can accurately describe both structured and disordered regions of such proteins. Using the special-purpose Anton 2 supercomputer, we benchmarked the efficacy of nine presently available molecular force fields in describing the structure and dynamics of a Fused in sarcoma (FUS) protein. Five-microsecond simulations of the full-length FUS protein characterized the effect of the force field on the global conformation of the protein, self-interactions among its side chains, solvent accessible surface area, and the diffusion constant. Using the results of dynamic light scattering as a benchmark for the FUS radius of gyration, we identified several force fields that produced FUS conformations within the experimental range. Next, we used these force fields to perform ten-microsecond simulations of two structured RNA binding domains of FUS bound to their respective RNA targets, finding the choice of the force field to affect stability of the RNA-FUS complex. Taken together, our data suggest that a combination of protein and RNA force fields sharing a common four-point water model provides an optimal description of proteins containing both disordered and structured regions and RNA-protein interactions. To make simulations of such systems available beyond the Anton 2 machines, we describe and validate implementation of the best performing force fields in a publicly available molecular dynamics program NAMD. Our NAMD implementation enables simulations of large (tens of millions of atoms) biological condensate systems and makes such simulations accessible to a broader scientific community.

Phenotypic analysis of HIV-1 resistance to CD4 binding site broadly-neutralizing antibodies

Abstract Broadly neutralizing antibodies (bNAbs) show strong immunotherapy potential by targeting conserved regions of the HIV-1 envelope. Single bNAb use in viremic individuals quickly results in the emergence of escape variants, likely originating from existing quasispecies. An ex vivo assay was developed to predict bNAb efficacy in viremic individuals. The development of a predictive bNAb efficacy model may assist with the planning of future clinical trials. The ex vivoassay used pre-infusion CD4 +T cells from participants enrolled in the VRC 607/ACTG A5378 phase I study of the CD4 binding site (CD4bs) bNAbs’ VRC01LS (n=7) or VRC07-523LS (n=9). From these assays, the escape variant signatures of outgrowth viruses that replicated in presence of CD4bs bNAbs, were identified. We found that these escape variants contained key changes to the ß23-V5 region of the gp120 envelope, including charge changes, glycan loss, and/or glycan shifts. Outgrowth viruses from each participant had similar escape mutation patterns. Resistance mutations of the viral env sequences were confirmed in pseudovirus-based neutralization assays. We found complementary escape profiles from 3 CD4bs bNAbs (1–18LS, N6LS, and VRC01v23) with ex vivoand neutralization assays showing suppression by 1 or 2 but not all 3 bNAbs, supporting the potential for combination bNAb therapy. Our data suggest that the simultaneous use of multiple CD4 binding site bNAbs together could aid in limiting viral escape. Supported by intramural funds from NIH.

In use of Olaparib for ovarian cancer: understanding from pathogenesis, target corroboration, discovering process and treatment techanism

The second most prevalent gynecologic cancer, ovarian cancer has a poor prognosis, is difficult to diagnose, and has a high mortality. EOC (epithelial ovarian cancer) is the most common subtype. PARP inhibitor has so far been approved by FDA, and used for target therapy of epithelial ovarian cancer worldwide. This article based on lots of literature research, reviews studies of ovarian cancer pathogenesis, target corroboration, Olaparib discovering process and treatment mechanism. Somatic sequence mutation occupies a prominent position in the pathogenesis of ovarian cancer. Type ovarian cancer behave PIK3CA KRAS, BRAF, PTEN, and CTNNB1 sequence mutation. Type ovarian cancers exhibit TP53 and BRCA1/2 sequence mutation. Angiogenesis, TP53, and PARP are studies in target therapy of ovarian cancer. PARP is regarded as ideal target, Olaparib is the first PARP inhibitors. Preventing PARP works in DNA damage repairing in BRCA mutation ovarian cancer cells causing cell death preferentially are confirmed as the therapeutic mechanism of Olaparib (PARP inhibitor), in therapy of ovarian cancer.

Modeling SARS-CoV-2 nucleotide mutations as a stochastic process.

This study analyzes the SARS-CoV-2 genome sequence mutations by modeling its nucleotide mutations as a stochastic process in both the time-series and spatial domain of the gene sequence. In the time-series model, a Markov Chain embedded Poisson random process characterizes the mutation rate matrix, while the spatial gene sequence model delineates the distribution of mutation inter-occurrence distances. Our experiment focuses on five key variants of concern that had become a global concern due to their high transmissibility and virulence. The time-series results reveal distinct asymmetries in mutation rate and propensities among different nucleotides and across different strains, with a mean mutation rate of approximately 2 mutations per month. In particular, our spatial gene sequence results reveal some novel biological insights on the characteristic distribution of mutation inter-occurrence distances, which display a notable pattern similar to other natural diseases. Our findings contribute interesting insights to the underlying biological mechanism of SARS-CoV-2 mutations, bringing us one step closer to improving the accuracy of existing mutation prediction models. This research could also potentially pave the way for future work in adopting similar spatial random process models and advanced spatial pattern recognition algorithms in order to characterize mutations on other different kinds of virus families.

Neuropilin-1 Knockout and Rescue Confirms Its Role to Promote Metastasis in MDA-MB-231 Breast Cancer Cells.

Breast cancer (BC) metastasis remains a leading cause of female mortality. Neuropilin-1 (NRP-1) is a glycoprotein receptor that plays ligand-dependent roles in BC. Clinical studies indicate its correlation with metastatic disease; however, its functional role in BC metastasis remains uncertain. CRISPR-Cas9 was used to knockout the NRP-1 gene in MDA-MB-231 BC cells, and the effects on metastasis were determined using an orthotopic mouse engraftment model. NRP-1 expression in knockout cells was rescued using a recombinant cDNA with a silent mutation in the sgRNA target-adjacent PAM sequence. Differentially expressed genes between NRP-1 knockout and control cells were determined using whole-transcriptome sequencing and validated using real-time PCR. NRP-1KO cells showed a pronounced reduction in the metastasis to the lungs. KEGG pathway analysis of the transcriptome data revealed that PI3K and ECM receptor interactions were among the top altered pathways in the NRP-1KO cells. In addition, reduction in metastasis enhancers proteins, Integrin-β3 and Tenascin-C, and genes CCL20 and FN1 and upregulation of metastasis suppressor genes, ACVRL and GPX3 in NRP-1KO were detected. These findings provide evidence for a functional role for NRP-1 in BC metastasis, supporting further exploration of NRP-1 and the identified genes as targets in treating metastatic BC.

Tackling the Pangenome Dilemma Requires the Concerted Analysis of Multiple Population Genetic Processes.

The pangenome is the set of all genes present in a prokaryotic population. Most pangenomes contain many accessory genes of low and intermediate frequencies. Different population genetics processes contribute to the shape of these pangenomes, namely selection and fitness-independent processes such as gene transfer, gene loss, and migration. However, their relative importance is unknown and highly debated. Here, we argue that the debate around prokaryotic pangenomes arose due to the imprecise application of population genetics models. Most importantly, two different processes of horizontal gene transfer act on prokaryotic populations, which are frequently confused, despite their fundamentally different behavior. Genes acquired from distantly related organisms (termed here acquiring gene transfer) are most comparable to mutation in nucleotide sequences. In contrast, gene gain within the population (termed here spreading gene transfer) has an effect on gene frequencies that is identical to the effect of positive selection on single genes. We thus show that selection and fitness-independent population genetic processes affecting pangenomes are indistinguishable at the level of single gene dynamics. Nevertheless, population genetics processes are fundamentally different when considering the joint distribution of all accessory genes across individuals of a population. We propose that, to understand to which degree the different processes shaped pangenome diversity, the development of comprehensive models and simulation tools is mandatory. Furthermore, we need to identify summary statistics and measurable features that can distinguish between the processes, where considering the joint distribution of accessory genes across individuals of a population will be particularly relevant.