Stable Expression Research Articles

Screening of reference genes for gene expression study in different tissues from the transcriptome data of the vector leafhopper Psammotettix striatus

Selecting appropriate reference genes is crucial for ensuring the accuracy and reliability of gene expression study using reverse transcription-quantitative PCR (RT-qPCR). To screen the optimal reference genes for analyzing gene expression in different tissues of the vector leafhopper Psammotettix striatus which causes extensive damage to a wide range of crops by vectoring multiple plant pathogenic microorganisms, the transcriptome data from Malpighian tubules (MTs) of P. striatus were mined. Twenty alternative candidate reference genes were initially selected for screening, among which seven genes with diverse Gene Ontology (GO) annotations were choosed as candidate reference genes, i.e., ribosomal protein L7A (RPL7A), ribosomal protein S28 (RPS28), ribosomal protein L22 (RPL22), ribosomal protein LP2 (RPLP2), H3 histone family 3A (H3F3A), elongation factor 1γ (EF-1γ), and elongation factor 1α (EF-1α). Gene expression levels in different tissues of P. striatus adults were examined using RT-qPCR, and their expression stability was analyzed using multiple reference gene screening software. This study revealed EF-1α as the most abundantly expressed gene, while RPL22 exhibited the lowest expression levels. EF-1α showed the most stable expression, whereas RPS28 showed the least stability. Various software tools confirmed EF-1α as the most stable single reference gene, and EF-1α and RPLP2 an optimal combination. This study provides a foundation for future investigation of the transmission of pathogenic microorganisms mediated by the vector leafhoppers, the function of the MTs, the biosynthesis of brochosomes, the coevolutionary processes and nutritional interactions of symbionts and host insects, and the gene expression study of other sap-sucking insects.

Immune protection of grass carp by oral vaccination with recombinant Bacillus methylotrophicus expressing the heterologous tolC gene

In the field of aquaculture, the enhancement of animal health and disease prevention is progressively being tackled using alternatives to antibiotics, including vaccines and probiotics. This study was designed to evaluate the potential of a recombinant Bacillus methylotrophicus, engineered to express the outer membrane channel protein TolC of Aeromonas hydrophila AH3 and the green fluorescent protein GFP, as an oral vaccine. Initially, the genes encoding tolC and GFP were cloned into a prokaryotic expression system, and anti-TolC mouse antiserum was generated. Subsequently, the tolC gene was subcloned into a modified pMDGFP plasmid, which was transformed into B. methylotrophicus WM-1 for protein expression. The recombinant B. methylotrophicus BmT was then administered to grass carp via co-feeding, and its efficacy as an oral vaccine was assessed. Our findings demonstrated successful expression of the 55 kDa TolC and 28 kDa GFP proteins, and the preparation of polyclonal antibodies with high specificity. The BmT exhibited stable expression of the GFP-TolC fusion protein and excellent genetic stability. Following oral immunization, significant elevations were observed in serum-specific IgM levels and the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) in grass carp. Concurrently, significant upregulation of immune-related genes, including IFN-I, IL-10, IL-1β, TNF-α, and IgT, was noted in the intestines, head kidney, and spleen of the grass carp. Colonization tests further revealed that the BmT persisted in the gut of immunized fish even after a fasting period of 7 days. Notably, oral administration of BmT enhanced the survival rate of grass carp following A. hydrophila infection. These results suggest that the oral BmT vaccine developed in this study holds promise for future applications in aquaculture.

Patient derived cancer organoids model the response to HER2-CD3 bispecific antibody (BsAbHER2) generated from hydroxyapatite gene delivery system

HER2-positive cancer is a prevalent subtype of malignancy with poor prognosis, yet current targeted therapies, like Trastuzumab and pyrotinib, have resulted in remission in patients with HER2-positive cancer. This study provides a novel approach for immunotherapy based on a hydroxyapatite (HA) gene delivery system producing a bispecific antibody for HER2-positive cancer treatment. An HA nanocarrier has been synthesized by the classical hydrothermal method. Particularly, the HA-nanoneedle system was able to mediate stable gene expression of minicircle DNA (MC) encoding a humanized anti-CD3/anti-HER2 bispecific antibody (BsAbHER2) in vivo. The produced BsAbs exhibited a potent killing effect not only in HER2-positive cancer cells but also in patient-derived organoids in vitro. This HA-nanoneedle gene delivery system features simple large-scale preparation and clinical applicability. Hence, the HA-nanoneedle gene delivery system combined with minicircle DNA vector encoding BsAbHER2 reported here provides a potential immunotherapy strategy for HER2-positive tumors.

Selective degradation of hyperphosphorylated tau by proteolysis-targeting chimeras ameliorates cognitive function in Alzheimer's disease model mice.

Alzheimer's disease (AD) is one of the most common chronic neurodegenerative diseases. Hyperphosphorylated tau plays an indispensable role in neuronal dysfunction and synaptic damage in AD. Proteolysis-targeting chimeras (PROTACs) are a novel type of chimeric molecule that can degrade target proteins by inducing their polyubiquitination. This approach has shown promise for reducing tau protein levels, which is a potential therapeutic target for AD. Compared with traditional drug therapies, the use of PROTACs to reduce tau levels may offer a more specific and efficient strategy for treating AD, with fewer side effects. In the present study, we designed and synthesized a series of small-molecule PROTACs to knock down tau protein. Of these, compound C8 was able to lower both total and phosphorylated tau levels in HEK293 cells with stable expression of wild-type full-length human tau (termed HEK293-htau) and htau-overexpressed mice. Western blot findings indicated that C8 degraded tau protein through the ubiquitin-proteasome system in a time-dependent manner. In htau-overexpressed mice, the results of both the novel object recognition and Morris water maze tests revealed that C8 markedly improved cognitive function. Together, our findings suggest that the use of the small-molecule PROTAC C8 to degrade phosphorylated tau may be a promising therapeutic strategy for AD.

Systematic selection of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Lutjanuserythropterus

Quantitative real-time PCR (qRT-PCR) has been widely employed for the study of gene expression in fish, and accurate normalization is crucial. In this study, we aimed to identify the most stably expressed genes in various tissues, different developmental stages, and within astaxanthin treatment groups in Lutjanuserythropterus. Twelve candidate genes (EEF1A, CYB5R3, DLD, IDH3A, MRPL17, MRPL43, NDUFS7, PABPC1, PAGR1, PFDN2, PSMC3, and RAB10) were examined via qRT-PCR. We employed geNorm and NormFinder to assess their stability. The results revealed that RAB10 and PFDN2 exhibited relatively stable expression patterns across different tissue and astaxanthin treatment groups, while NDUFS7 and MRPL17 proved to be the most reliable reference gene combinations across various developmental stages. The stability of these selected genes was further validated by assessing the expression of two target genes, CRADD and CAPNS1, across developmental stages, reinforcing the reliability of NDUFS7 as it closely aligned with transcriptome-wide expression patterns at these stages. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in L.erythropterus.

AAVS1‐targeted, stable expression of ChR2 in human brain organoids for consistent optogenetic control

AbstractSelf‐organizing brain organoids provide a promising tool for studying human development and disease. Here we created human forebrain organoids with stable and homogeneous expression of channelrhodopsin‐2 (ChR2) by generating AAVS1 safe harbor locus‐targeted, ChR2 knocked‐in human pluripotent stem cells (hPSCs), followed by the differentiation of these genetically engineered hPSCs into forebrain organoids. The resulting ChR2‐expressing human forebrain organoids showed homogeneous cellular expression of ChR2 throughout entire regions without any structural and functional perturbations and displayed consistent and robust neural activation upon light stimulation, allowing for the non‐virus mediated, spatiotemporal optogenetic control of neural activities. Furthermore, in the hybrid platform in which brain organoids are connected with spinal cord organoids and skeletal muscle spheroids, ChR2 knocked‐in forebrain organoids induced strong and consistent muscle contraction upon brain‐specific optogenetic stimulation. Our study thus provides a novel, non‐virus mediated, preclinical human organoid system for light‐inducible, consistent control of neural activities to study neural circuits and dynamics in normal and disease‐specific human brains as well as neural connections between brain and other peripheral tissues.

Selection and application of small non-coding RNAs for normalizing RT-qPCR data of bovine preimplantation embryo conditioned medium

Small non-coding RNAs (sncRNAs) present in the conditioned medium (CM) of bovine preimplantation embryos are potential noninvasive biomarkers for assessing embryo quality. Accurate quantification of sncRNA levels in the spent CM is of utmost importance in this regard. RT-qPCR is considered as the gold standard for quantifying RNA. In order to standardize RT-qPCR data in the sample type under investigation, the use of suitable stable sncRNAs is essential. Here, we selected 10 sncRNAs from small RNA sequencing of CM samples derived from both bovine blastocysts and degenerate embryos, and evaluated their expression stability together with that of cel-miR-39 as a spike and the often-used U6 small nuclear RNA at different embryo developmental stages. In CM of 2-cell embryos, rsRNA-1044 showed the most stable expression, while tDR-1:32-Gly–CCC–1 was the most stable expressed sncRNA in CM of the stages beyond the 2-cell stage. Next, tDR-1:32-Gly–CCC–1 was used for normalizing the RT-qPCR data from the CM of blastocysts and degenerate embryos. Bta-miR-155 and tDR-39:75-Arg-CCG-2 were found to be significantly up-regulated in the CM of blastocysts compared to that of the degenerated embryos (P = 0.028 and P = 0.017, respectively), suggesting their expression levels are related to embryo development stage. In conclusion, tDR-1:32-Gly–CCC–1 can serve as a suitable reference sncRNA for normalization of RT-qPCR data of the CM from bovine blastocysts.

Biomarker conversion from primary breast cancer to synchronous axillary lymph node metastasis and neoadjuvant therapy response: a single-center analysis

PurposeThe biomarker characteristics of breast cancer plays an important role in predicting treatment sensitivity. The aim of the present study was to compare immunohistochemical profiles (ER, PR, HER2, and Ki67) between the primary tumor and synchronous axillary lymph node metastasis and investigate the subsequent effects on neoadjuvant therapy response.MethodsA total of 358 patients with pathologically confirmed synchronous axillary lymph node metastasis at first diagnosis and treated by neoadjuvant therapy at Peking University First Hospital from January 1, 2013 to December 31, 2022 were included in this retrospective study. Clinicopathologic data, especially receptor status in primary and metastatic foci, was collected for each case.ResultsChange of ER, PR, HER2, and Ki67 expression was observed in 5.9%, 8.7%, 12.6%, and 17.3% of patients, respectively. HR discordance was observed more frequently when the ER status (p = 0.023) or PR status (p = 0.010) of primary tumor was negative, while HER2 discordance seemed to be more frequent when the HER2 status of primary tumor was HER2-0 or HER2-low (p < 0.001). Patients with loss of HR-positivity (positive to negative) responded to neoadjuvant chemotherapy better compared to those with stable positive HR expression (50% vs. 11.1%, p = 0.0017). A significantly decrease in pCR rate was observed in patients with unstable HER2 status, but not in the HER2-0/HER2-low subgroup.ConclusionReceptor discordance between primary tumor and synchronous axillary LNM appears to already exist before any anti-tumor therapy. This instability has limited clinical impact on the choice of neoadjuvant therapy at current stage, but further investigation is warranted with the incremental application of endocrine drugs and ADCs in neoadjuvant therapy.

Plasticity and lineage commitment of individual TH1 cells are determined by stable T-bet expression quantities.

T helper 1 (TH1) cell identity is defined by the expression of the lineage-specifying transcription factor T-bet. Here, we examine the influence of T-bet expression heterogeneity on subset plasticity by leveraging cell sorting of distinct in vivo-differentiated TH1 cells based on their quantitative expression of T-bet and interferon-γ. Heterogeneous T-bet expression states were regulated by virus-induced type I interferons and were stably maintained even after secondary viral infection. Exposed to alternative differentiation signals, the sorted subpopulations exhibited graded levels of plasticity, particularly toward the TH2 lineage: T-bet quantities were inversely correlated with the ability to express the TH2 lineage-specifying transcription factor GATA-3 and TH2 cytokines. Reprogramed TH1 cells acquired graded mixed TH1 + TH2 phenotypes with a hybrid epigenetic landscape. Continuous presence of T-bet in differentiated TH1 cells was essential to ensure TH1 cell stability. Thus, innate cytokine signals regulate TH1 cell plasticity via an individual cell-intrinsic rheostat to enable T cell subset adaptation to subsequent challenges.

Heart immunoengineering by lentiviral vector-mediated genetic modification during normothermic ex vivo perfusion.

Heart transplantation is associated with major hurdles, including the limited number of available organs for transplantation, the risk of rejection due to genetic discrepancies, and the burden of immunosuppression. In this study, we demonstrated the feasibility of permanent genetic engineering of the heart during ex vivo perfusion. Lentiviral vectors encoding for short hairpin RNAs targeting beta2-microglobulin (shβ2m) and class II transactivator (shCIITA) were delivered to the graft during two hours of normothermic EVHP. Highly efficient genetic engineering was indicated by stable reporter gene expression in endothelial cells and cardiomyocytes. Remarkably, swine leucocyte antigen (SLA) class I and SLA class II expression levels were decreased by 66% and 76%, respectively, in the vascular endothelium. Evaluation of lactate, troponin T, and LDH levels in the perfusate and histological analysis showed no additional cell injury or tissue damage caused by lentiviral vectors. Moreover, cytokine secretion profiles (IL-6, IL-8, and TNF-α) of non-transduced and lentiviral vector-transduced hearts were comparable. This study demonstrated the ex vivo generation of genetically engineered hearts without compromising tissue integrity. Downregulation of SLA expression may contribute to reduce the immunogenicity of the heart and support graft survival after allogeneic or xenogeneic transplantation.

Mitochondria-targeted catalase induced cell malignant transformation by the downregulation of p53 protein stability via USP28/miR-200b/PP2A-Cα axis

Antioxidants exert a paradoxical influence on cancer prevention. The latest explanation for this paradox is the different target sites of antioxidants. However, it remains unclear how mitochondria-targeted antioxidants trigger specific p53-dependent pathways in malignant transformation models. Our study revealed that overexpression of mitochondria-targeted catalase (mCAT) instigated such malignant transformation via mouse double minute 2 homolog (MDM2) -mediated p53 degradation. In mouse epithelial JB6 Cl41 cells, the stable expression of mCAT resulted in MDM2-mediated p53 degradation, unlike in catalase-overexpressed Cl41 cells. Further, we demonstrated that mCAT overexpression upregulated ubiquitin-specific protease 28 (USP28) expression, which in turn stabilized c-Jun protein levels. This alteration initiated the activation of the miR-200b promoter transcription activity and a subsequent increase in miR-200b expression. Furthermore, elevated miR-200b levels then promoted its binding to the 3′-untranslated region of protein phosphatase 2A catalytic subunit (PP2A-C) α-isoform mRNA, consequently resulting in PP2A-C protein downregulation. This cascade of events ultimately contributed to increased MDM2 phosphorylation and p53 protein degradation. Thus, the mCAT overexpression triggers MDM2/p53-dependent malignant transformation through USP28/miR-200b/PP2A-Cα pathway, which may provide a new information for understanding mitochondria-targeted antioxidants facilitate the progression to the tumorigenic state.

Intersection of the lexical-semantic fields “Mentality” and “Physiology” in the poetic language of Igor-Severyanin

The intersection of two lexical-semantic fields “mentality” and “physiology” in the poetic texts of Igor-Severyanin is studied. The relevance of the research is determined by the fact that within the framework of modern anthropocentric approach and increased attention to the mental sphere lexical semantics is investigated, lexical-semantic fields, their intersections, semantic shifts are considered, actual problems of linguistic poetics are touched upon. The aim of the work is to explore the intersection of lexical-semantic fields “mentality” and “physiology” in the language of Severyanin's poems. The material includes lexemes of the mental sphere from Igor-Severyanin's poetic texts. While determining the usual meaning of these lexemes, the authors used the materials of “Explanatory Dictionary of the Russian Language” by S.I. Ozhegov. The methods of description, comparison, contrast, lexical-semantic and contextual analysis, and continuous sampling were applied; the lexical-semantic field - a generally accepted form of representing lexical units by their meaning - was used to structure the vocabulary. The intersections of the lexical-semantic fields “mentality” and “physiology” in Igor-Severyanin's texts, revealed in the research, are primarily due to the inseparable connection between the inner world of man and his bodily organs of sense. The language fixes stable expressions indicating the possibility of comprehending the human consciousness through analyzing the signals of his body. Words used to denote phenomena and processes related to the body, health, gestures, etc., are reinterpreted in poetic language and receive new semantics, which allows to refer these new lexical-semantic units to the lexical-semantic field of mentality. In Igor-Severyanin's poems the inner world of the lyrical subject dominates, which is reflected in the author's word usage. Even words and expressions with the semantics of physiology shift in their meanings, acquiring the semantics of mentality, at the same time thoughts and feelings are endowed with the author's positive evaluation. The prospects of the research are connected primarily with further study of the mental sphere in the Russian linguistic lexical-semantic system, its dynamics, as well as with the analysis of semantic shifts in the poetic language vocabulary.

Lysophosphatidylcholine acyltransferase 1 suppresses nanoclustering and function of KRAS.

KRAS is frequently mutated in cancer, contributing to 20% of all human cancer especially pancreatic, colorectal and lung cancer. Signaling of the constitutively active KRAS oncogenic mutants is mostly compartmentalized to proteolipid nanoclusters on the plasma membrane (PM). Signaling nanoclusters of many KRAS mutants selectively enrich phosphatidylserine (PS) lipids with unsaturated sn-2 acyl chains, but not the fully saturated PS species. Thus, remodeling PS acyl chains may suppress KRAS oncogenesis. Lysophosphatidylcholine acyltransferases (LPCATs) remodel sn-2 acyl chains of phospholipids, with LPCAT1 preferentially generating the fully saturated lipids. Here, we show that stable expression of LPCAT1 depletes major PS species with unsaturated sn-2 chains while decreasing minor phosphatidylcholine (PC) species with the corresponding acyl chains. LPCAT1 expression more effectively disrupts the nanoclustering of oncogenic GFP-KRASG12V, which is restored by acute addback of exogenous unsaturated PS. LPCAT1 expression compromises signaling and oncogenic activities of the KRAS-dependent pancreatic tumor lines. LPCAT1 expression sensitizes human pancreatic tumor MiaPaCa-2 cells to KRASG12C specific inhibitor, Sotorasib. Statistical analyses of patient data further reveal that pancreatic cancer patients with KRAS mutations express less LPCAT1. Higher LPCAT1 expression also improves survival probability of pancreatic and lung adenocarcinoma patients with KRAS mutations. Thus, PS acyl chain remodeling selectively suppresses KRAS oncogenesis.

Purification of Adeno-Associated Viral Vector Serotype 9 Using Ceramic Hydroxyapatite Chromatography and its Analysis.

Adeno-associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissues in vivo. Owing to their convenience, high efficiency, long-term stable gene expression, and minimal side effects, AAV vectors have become one of the gold standards for investigating gene functions in vivo, especially in non-clinical studies. However, challenges persist in efficiently preparing a substantial quantity of high-quality AAV vectors. Commercial AAV vectors are typically associated with high costs. Further, in-laboratory production is hindered by the lack of specific laboratory equipment, such as ultracentrifuges. Therefore, a simple, quick, and scalable preparation method for AAV vectors is needed for proof-of-concept experiments. Herein, we present an optimized method for producing and purifying high-quality AAV serotype 9 (AAV9) vectors using standard laboratory equipment and chromatography. Using ceramic hydroxyapatite as a mixed-mode chromatography medium can markedly increase the quality of purified AAV vectors. Basic Protocols and optional methods for evaluating purified AAV vectors are also described. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of AAV9 vectors in 293EB cells Basic Protocol 2: Concentration and buffer exchange of AAV9 vectors from 293EB cell culture supernatants using tangential flow filtration Basic Protocol 3: Purification of AAV9 vectors from TFF samples using ceramic hydroxyapatite chromatography Basic Protocol 4: Analysis of the purified AAV9 vectors.

Rapid production of COVID-19 subunit vaccine candidates and their immunogenicity evaluation in pigs

The emergence of various variants of concern (VOCs) necessitates the development of more efficient vaccines for COVID-19. In this study, we established a rapid and robust production platform for a novel subunit vaccine candidate based on eukaryotic HEK-293 T cells. The immunogenicity of the vaccine candidate was evaluated in pigs. The results demonstrated that the pseudovirus neutralizing antibody (pNAb) titers reached 7751 and 306 for the SARS-CoV-2 Delta and Omicron variants, respectively, after the first boost. Subsequently, pNAb titers further increased to 10,201 and 1350, respectively, after the second boost. Additionally, ELISPOT analysis revealed a robust T-cell response characterized by IFN-γ (171 SFCs/106 cells) and IL-2 (101 SFCs/106 cells) production. Our study demonstrates that a vaccine candidate based on the Delta variant spike protein may provide strong and broad protection against the prototype SARS-CoV-2 and VOCs. Moreover, the strategy for the efficient and stable expression of recombinant proteins utilizing HEK-293 T cells can be employed as a universal platform for future vaccine development.

Atoh1 overexpression promotes Guinea pig bone marrow mesenchymal stem cells to differentiate into neural stem cell

Sensorineural hearing loss (SNHL) is a prevalent condition in otolaryngology. A key obstacle is finding effective strategies for regenerating damaged cochlear hair cells in adult animals. A practical and reliable approach has been developed to create a superior cell source for stem cell transplantation in the inner ear to treat SNHL. Atoh1 is involved in the differentiation of neurons, intestinal secretory cells, and mechanoreceptors including auditory hair cells, and thus plays an important role in neurogenesis. Lentivirus-mediated transfection of bone marrow mesenchymal stem cells (BMSCs) was utilized to achieve stable expression of the essential transcription factor Atoh1, which is crucial for developing auditory hair cells without compromising cell survival. By manipulating the induction conditions through altering the cell growth environment using anti-adherent culture, the synergistic impact of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) was effectively applied to significantly improve the differentiation efficiency of bone marrow-derived mesenchymal stem cells (BMSC) into neural stem cells (NSCs) following Atoh1 transfection, thereby reducing the induction time. The study indicated that the newly proposed transdifferentiation method effectively transformed BMSCs into NSCs in a controlled environment, presenting a potential approach for stem cell transplantation to promote hair cell regeneration.

Housekeeping protein-coding genes interrogated with tissue and individual variations

Housekeeping protein-coding genes are stably expressed genes in cells and tissues that are thought to be engaged in fundamental cellular biological functions. They are often utilized as normalization references in molecular biology research and are especially important in integrated bioinformatic investigations. Prior studies have examined human housekeeping protein-coding genes by analyzing various gene expression datasets. The inclusion of different tissue types significantly impacted the discovery of housekeeping genes. In this report, we investigated particularly individual human subject expression differences in protein-coding genes across different tissue types. We used GTEx V8 gene expression datasets obtained from more than 16,000 human normal tissue samples. Furthermore, the Gini index is utilized to investigate the expression variations of protein-coding genes between tissue and individual donor subjects. Housekeeping protein-coding genes found using Gini index profiles may vary depending on the tissue subtypes investigated, particularly given the diverse sample size collections across the GTEx tissue subtypes. We subsequently selected major tissues and identified subsets of housekeeping genes with stable expression levels among human donors within those tissues. In this work, we provide alternative sets of housekeeping protein-coding genes that show more consistent expression patterns in human subjects across major solid organs. Weblink: https://hpsv.ibms.sinica.edu.tw.

Seeing double: two different homospermidine oxidases are involved in pyrrolizidine alkaloid biosynthesis in different organs of comfrey (Symphytum officinale).

Pyrrolizidine alkaloids (PAs) are toxic specialized metabolites produced in several plant species and frequently contaminate herbal teas or livestock feed. In comfrey (Symphytum officinale, Boraginaceae), they are produced in two different organs of the plant, the root and young leaves. In this study, we demonstrate that homospermidine oxidase (HSO), a copper-containing amine oxidase (CuAO) responsible for catalyzing the formation of the distinctive pyrrolizidine ring in PAs, is encoded by two individual genes. Specifically, SoCuAO1 is expressed in young leaves, while SoCuAO5 is expressed in roots. CRISPR/Cas9-mediated knockout of socuao5 resulted in hairy roots (HRs) unable to produce PAs, supporting its function as HSO in roots. Plants regenerated from socuao5 knockout HRs remained completely PA-free until the plants began to develop inflorescences, indicating the presence of another HSO that is expressed only during flower development. Stable expression of SoCuAO1 in socuao5 knockout HRs rescued the ability to produce PAs. In vitro assays of both enzymes transiently expressed in Nicotiana benthamiana confirmed their HSO activity and revealed the ability of HSO to control the stereospecific cyclization of the pyrrolizidine backbone. The observation that the first specific step of PA biosynthesis catalyzed by homospermidine synthase requires only one gene copy, while two independent paralogs are recruited for the subsequent homospermidine oxidation in different tissues of the plant, suggests a complex regulation of the pathway. This adds a new level of complexity to PA biosynthesis, a system already characterized by species-specific, tight spatio-temporal regulation, and independent evolutionary origins in multiple plant lineages.

CRISPR-Cas9 immune-evasive hESCs are rejected following transplantation into immunocompetent mice.

Although current stem cell therapies exhibit promising potential, the extended process of employing autologous cells and the necessity for donor-host matching to avert the rejection of transplanted cells significantly limit the widespread applicability of these treatments. It would be highly advantageous to generate a pluripotent universal donor stem cell line that is immune-evasive and, therefore, not restricted by the individual's immune system, enabling unlimited application within cell replacement therapies. Before such immune-evasive stem cells can be moved forward to clinical trials, in vivo testing via transplantation experiments in immune-competent animals would be a favorable approach preceding preclinical testing. By using human stem cells in immune competent animals, results will be more translatable to a clinical setting, as no parts of the immune system have been altered, although in a xenogeneic setting. In this way, immune evasiveness, cell survival, and unwanted proliferative effects can be assessed before clinical trials in humans. The current study presents the generation and characterization of three human embryonic stem cell lines (hESCs) for xenogeneic transplantation in immune-competent mice. The major histocompatibility complexes I- and II-encoding genes, B2M and CIITA, have been deleted from the hESCs using CRISPR-Cas9-targeted gene replacement strategies and knockout. B2M was knocked out by the insertion of murine CD47. Human-secreted embryonic alkaline phosphatase (hSEAP) was inserted in a safe harbor site to track cells in vivo. The edited hESCs maintained their pluripotency, karyotypic normality, and stable expression of murine CD47 and hSEAP in vitro. In vivo transplantation of hESCs into immune-competent BALB/c mice was successfully monitored by measuring hSEAP in blood samples. Nevertheless, transplantation of immune-evasive hESCs resulted in complete rejection within 11days, with clear immune infiltration of T-cells on day 8. Our results reveal that knockout of B2M and CIITA together with species-specific expression of CD47 are insufficient to prevent rejection in an immune-competent and xenogeneic context.

Development of a chloroplast expression system for Dunaliella salina

Dunaliella salina is an innovative expression system due to its distinct advantages such as high salt tolerance, low susceptibility to contamination, and the absence of the cell wall. While nuclear transformation has been extensively studied, research on D. salina chloroplast transformation remains in the preliminary stages. In this study, we established an efficient chloroplast expression system for D. salina using Golden Gate assembly. We developed a D. salina toolkit comprising essential components such as chloroplast-specific promoters, terminators, homologous fragments, and various vectors. We confirmed its functionality by expressing the EGFP protein. Moreover, we detailed the methodology of the entire construction process. This expression system enables the specific targeting of foreign genes through simple homologous recombination, resulting in stable expression in chloroplasts. The toolkit achieved a relatively high transformation efficiency within a shorter experimental cycle. Consequently, the construction and utilization of this toolkit have the potential to enhance the efficiency of transgenic engineering in D. salina and advance the development of microalgal biofactories.