Wild-type Research Articles

Genome-wide analysis of tobacco NtTOM1/TOM3 gene family and identification of NtTOM1a/ NtTOM3a response to tobacco mosaic virus

BackgroundTOBAMOVIRUS MULTIPLICATION 1 (TOM1) and its homolog TOBAMOVIRUS MULTIPLICATION 3 (TOM3) play a prominent role in the multiplication of tobacco mosaic virus (TMV) in higher plants. Although homologs of NtTOM1/TOM3 genes have been identified in several plant species, little is known about the characteristics and functions of NtTOM1/TOM3 at the genome-wide level in tobacco (Nicotiana tabacum L.).ResultsIn this study, we performed genome-wide identification and expression pattern analysis of the tobacco NtTOM1/TOM3 gene family. Twelve NtTOM1/TOM3 genes were identified and classified into four groups based on phylogenetic analysis. Sequence and conserved domain analyses showed that all these genes contained a specific DUF1084 domain. Expression pattern analysis showed that NtTOM1a, NtTOM1b, NtTOM1d, NtTOM3a, NtTOM3b, and NtTOM3d were induced by TMV at 1-, 3-, and 9 dpi, whereas the expression of other genes was not responsive to TMV at the early infection stage. TMV virion accumulation showed no obvious difference in either nttom1a or nttom3a mutants compared with the wild type. However, the virus propagation was significantly, but not completely, inhibited in the nttom1atom3a double mutant, indicating that other gene family members may function redundantly, such as NtTOM1b and NtTOM1d. In addition, overexpression of NtTOM1a or NtTOM3a also inhibited the TMV replication to some extent.ConclusionsThe present study performed genome-wide analysis of the NtTOM1/TOM3 gene family in tobacco, and identified NtTOM1a and NtTOM3a as important genes involved in TMV multiplication based on functional analysis. These results provide a theoretical basis for further improving TMV resistance in tobacco.

Identification and analysis of differentially expressed lncRNAs and their ceRNA networks in DMD/mdx primary myoblasts

AbstractThis study explored the significance of long non-coding RNAs (lncRNAs), particularly their role in maintaining dystrophin protein stability and regulating myocyte proliferation and differentiation. The investigation focused on DMD/mdx mouse skeletal muscle primary myoblasts, aiming to identify lncRNAs potential as biomarkers and therapeutic targets for Duchenne muscular dystrophy (DMD). Utilizing CLC Genomics Workbench software, 554 differentially expressed lncRNAs were identified in DMD/mdx mice compared to wild-type (WT) control. Among them, 373 were upregulated, and 181 were downregulated. The study highlighted specific lncRNAs (e.g., 5930430L01Rik, Gm10143, LncRNA1490, LncRNA580) and their potential regulatory roles in DMD key genes like IGF1, FN1, TNNI1, and MYOD1. By predicting miRNA and their connections with lncRNA and mRNA (ceRNA network) using tools such as miRNet, miRSYSTEM and miRCARTA, the study revealed potential indirect regulation of Dystrophin, IGF1R and UTRN genes by identified lncRNAs (e.g. 2310001H17Rik-203, C130073E24Rik-202, LncRNA2767, 5930430L01Rik and LncRNA580). These findings suggest that the identified lncRNAs may play crucial roles in the development and progression of DMD through their regulatory influence on key gene expression, providing valuable insights for potential therapeutic interventions.

Rational Design of Formate Dehydrogenase for Enhanced Thermal Stability and Catalytic Activity in Bioelectrocatalysis.

Formate dehydrogenase can be utilized as a biocatalyst in the bioelectrocatalysis of converting CO2 into formic acid. However, its industrial application has been hindered by limited thermal stability. This study successfully obtained a mutant (D533S/E684I) with enhanced thermal stability and catalytic activity through the rational design of flexible regions. The mutant exhibited a half-life (t1/2) 1.5 times longer than the wild type (WT) at 35 °C, along with a specific enzyme activity 7.46 times higher than that of the WT. Additionally, the catalytic efficiency (kcat/Km value) of the mutant toward the substrate was 2.72 s-1·mM-1, representing a 19.4-fold increase compared to the WT (0.14 s-1·mM-1). Formic acid production reached 53.4 mM through bioelectrocatalysis after 10 h, utilizing the mutant as the biocatalyst. Molecular dynamics simulations and structural analysis were employed to investigate the molecular mechanisms behind the enhanced thermal stability and activity. The displacement of a highly flexible region in the mutant may counteract the stability-activity trade-off. This study proposed a method for improving both thermal stability and activity in enzyme evolution.

Autophagy controls neuronal differentiation by regulating the WNT-DVL signaling pathway.

Macroautophagy/autophagy dysregulation is associated with various neurological diseases, including Vici syndrome. We aimed to determine the role of autophagy in early brain development. We generated neurons from human embryonic stem cells and developed a Vici syndrome model by introducing a loss-of-function mutation in the EPG5 gene. Autophagy-related genes were upregulated at the neuronal progenitor cell stage. Inhibition of autolysosome formation with bafilomycin A1 treatment at the neuronal progenitor cell stage delayed neuronal differentiation. Notably, WNT (Wnt family member) signaling may be part of the underlying mechanism, which is negatively regulated by autophagy-mediated DVL2 (disheveled segment polarity protein 2) degradation. Disruption of autolysosome formation may lead to failure in the downregulation of WNT signaling, delaying neuronal differentiation. EPG5 mutations disturbed autolysosome formation, subsequently inducing defects in progenitor cell differentiation and cortical layer generation in organoids. Disrupted autophagy leads to smaller organoids, recapitulating Vici syndrome-associated microcephaly, and validating the disease relevance of our study.Abbreviations: BafA1: bafilomycin A1; co-IP: co-immunoprecipitation; DVL2: dishevelled segment polarity protein 2; EPG5: ectopic P-granules 5 autophagy tethering factor; gRNA, guide RNA; hESC: human embryonic stem cells; KO: knockout; mDA, midbrain dopamine; NIM: neural induction media; NPC: neuronal progenitor cell; qPCR: quantitative polymerase chain reaction; UPS: ubiquitin-proteasome system; WNT: Wnt family member; WT: wild type.

Modulation of the Arabidopsis Starch Metabolic Network by the Cytosolic Acetyl-CoA Pathway in the Context of the Diurnal Illumination Cycle

The starch metabolic network was investigated in relation to other metabolic processes by examining a mutant with altered single-gene expression of ATP citrate lyase (ACL), an enzyme responsible for generating cytosolic acetyl-CoA pool from citrate. Previous research has shown that transgenic antisense plants with reduced ACL activity accumulate abnormally enlarged starch granules. In this study, we explored the underlying molecular mechanisms linking cytosolic acetyl-CoA generation and starch metabolism under short-day photoperiods. We performed transcriptome and quantification of starch accumulation in the leaves of wild-type and antisense seedlings with reduced ACL activity. The antisense-ACLA mutant accumulated more starch than the wild type under short-day conditions. Zymogram analyses were conducted to compare the activities of starch-metabolizing enzymes with transcriptomic changes in the seedling. Differential expression between wild-type and antisense-ACLA plants was detected in genes implicated in starch and acetyl-CoA metabolism, and cell wall metabolism. These analyses revealed a strong correlation between the transcript levels of genes responsible for starch synthesis and degradation, reflecting coordinated regulation at the transcriptomic level. Furthermore, our data provide novel insights into the regulatory links between cytosolic acetyl-CoA metabolism and starch metabolic pathways.

Hydrogen peroxide participates in leaf senescence by inhibiting CHLI1 activity.

Hydrogen peroxide promoted leaf senescence by sulfenylating the magnesium chelating protease I subunit (CHLI1) in the chlorophyll synthesis pathway, and inhibited its activity to reduce chlorophyll synthesis. Leaf senescence is the final and crucial stage of plant growth and development, during which chlorophyll experiences varying degrees of destruction. It is well-known that the higher ROS accumulation is a key factor for leaf senescence, but whether and how ROS regulates chlorophyll synthesis in the process are unknown. Here, we report that H2O2 inhibits chlorophyll synthesis during leaf senescence via the I subunit of magnesium-chelatase (CHLI1). During leaf senescence, the decrease of chlorophyll content is accompanied by the increase of H2O2 accumulation, as well as the inhibition of catalase (CAT) genes expression. The mutant cat2-1, with increased H2O2 shows an accelerated senescence phenotype and decreased CHLI1 activity compared with the wild type. H2O2 inhibits CHLI1 activity by sulfenylating CHLI1 during leaf senescence. Consistent with this, the chli1 knockout mutant displays the same premature leaf senescence symptom as cat2-1, while overexpression of CHLI1 in cat2-1 can partially restore its early senescence phenotype. Taken together, these results illustrate that CAT2-mediated H2O2 accumulation during leaf senescence represses chlorophyll synthesis through sulfenylating CHLI1, and thus inhibits its activity, providing a new insight into the pivotal role of chlorophyll synthesis as a participant in orchestrating the leaf senescence.

A structure-based virtual screening identifies a novel MDM2 antagonist in the activation of the p53 signaling and inhibition of tumor growth.

p53, a tumor suppressor protein, has a vital role in the regulation of the cell cycle, apoptosis, and DNA damage repair. The degradation of p53 is predominantly controlled by the murine double minute 2 (MDM2) protein, a ubiquitin E3 ligase. The overexpression or amplification of MDM2 is commonly observed in various human cancers bearing wild-type p53 alleles, leading to the rapid degradation of the p53 protein and the attenuation of p53 tumor suppression functions. Thus, a major effort in p53-based cancer therapy has been to research MDM2 antagonists that specifically stabilize and activate p53, leading to the suppression of tumor growth. However, despite numerous efforts to develop MDM2 antagonists, to date they have failed to reach clinical use, largely because of the cytotoxicity associated with these small molecules. This study used our newly designed structure-based virtual screening approach on a commercial compound library to identify a novel compound, CGMA-Q18, which directly binds to MDM2, leading to the activation of p53, the induction of apoptosis, and cell cycle arrest in cancer cells. Notably, CGMA-Q18 significantly inhibited tumor xenograft growth in nude mice without observable toxicity. These findings highlight our useful virtual screening protocol and CGMA-Q18 as a putative MDM2 antagonist.

Molecular and Clinical Features of Pancreatic Acinar Cell Carcinoma: A Single-Institution Case Series

Objectives: Acinar cell carcinoma (ACC) accounts for about 1% of pancreatic cancers. The molecular and clinical features of ACC are less characterized than those of pancreatic ductal adenocarcinoma. Methods: We retrospectively evaluated the clinical and molecular features of ACC patients who underwent germline and/or somatic molecular testing at The University of Texas MD Anderson Cancer Center from 2008 to 2022 and two cases from 2023–2024 who underwent RNA and TME analysis by Boston Gene. Patient information was extracted from our institutional database with the approval of the Institutional Review Board. Results: We identified 16 patients with available molecular testing results. Fourteen patients had metastatic disease, one had borderline resectable disease, and one had localized resectable disease at diagnosis. Fifteen patients were wild type for KRAS (one patient had unknown KRAS status). Somatic/germline mutations of DNA damage repair genes (BRCA1/2, PALB2, and ATM) were present in 5 of 12 patients tested for these genes. One patient was found to have RET fusion and responded favorably to selpercatinib for over 42 months. The median overall survival (OS) was 24 months for patients with metastatic disease. One of the additional two cases who underwent BostonGene testing was found to have NTRK1 fusion. RNA and TME analysis by Boston Gene of the two cases reported immune desert features and relatively lower RNA levels of CEACAM5, CD47, CD74, and MMP1 and higher RNA levels of CDH6 compared with PDAC.

Stoichiometric Regeneration of Biomimetic Nicotinamide Coenzyme Powered by Biomass Sugars via In Vitro Synthetic Enzymatic Biosystems.

Biomimetic nicotinamide coenzymes, including nicotinamide mononucleotide (NMN+), have been demonstrated as promising low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) in biocatalysis. Herein, to efficiently regenerate NMNH from NMN+ in vitro powered by biomass sugars, a thermophilic NADP+-dependent glucose 6-phosphate dehydrogenase from Thermotoga maritima (TmG6PDH) was engineered to increase the activity toward NMN+. The catalytic efficiency (kcat/Km) of optimal mutant (TmG6PDH-R7) toward NMN+ increased by 71.7-fold than TmG6PDH-WT. As a result, compared to the wild type, the coenzyme specificity ([kcat/Km]NMN+/[kcat/Km]NADP+) of TmG6PDH-R7 increased by ~2.0×105-fold. The structural analysis revealed that the introduced hydrophobic and bulky residues lead to the formation of a smaller binding pocket, which resulting in a higher affinity for NMN+ with small size than NADP+. Then several in vitro synthetic enzymatic biosystems (ivSEBs) comprising this thermophilic TmG6PDH-R7 and a previously engineered thermophilic 6-phosphogluconate dehydrogenase were constructed. These ivSEBs harnessed the complete oxidation of renewable biomass sugars to facilitate the stoichiometric regeneration of 12 molecules of NMNH from 1 molecule of glucose, thereafter producing various products such as levodione, 2,3-butanediol or bioelectricity, over a wide temperature range. This study could pave the way for using stable and low-cost biomimetic coenzymes in ivSEBs for industrial biomanufacturing.

Genome-wide investigation of WRKY gene family in Lavandula angustifolia and potential role of LaWRKY57 and LaWRKY75 in the regulation of terpenoid biosynthesis

The WRKY transcription factors are integral to plant biology, serving essential functions in growth, development, stress responses, and the control of secondary metabolism. Through the use of bioinformatics techniques, this research has effectively characterized 207 members of the WRKY family (LaWRKY) present in the entire genome of Lavandula angustifolia. Phylogenetic analysis classified LaWRKYs into three distinct categories based on conserved domains. Collinearity analysis revealed tandem repeats, segmental duplications, and whole genome duplications in LaWRKYs, especially for segmental duplication playing a significant role in gene family expansion. LaWRKYs displayed distinct tissue-specific expression profiles across six different tissues of L. angustifolia. Particularly noteworthy were 12 genes exhibiting high expression in flower buds and calyx, the primary sites of volatile terpenoid production, indicating their potential role in terpenoid biosynthesis in L. angustifolia. RT-qPCR analysis revealed a notable increase in the expression levels of most examined LaWRKY genes in flower buds in response to both intense light and low-temperature conditions, whereas the majority of these genes in leaves were primarily induced by drought stress. However, all genes exhibited downregulation following GA treatment in both flower buds and leaves. Overexpression of LaWRKY57 (La13G01665) and LaWRKY75 (La16G00030) in tobacco led to a reduction in the density of glandular trichomes on leaf surfaces, resulting in changes to the volatile terpenoid composition in the leaves. Specifically, the content of Neophytadiene was significantly elevated compared to wild-type tobacco, while compounds such as eucalyptol, cis-3-Hexenyl iso-butyrate, and D-Limonene were produced, which were absent in the wild type. These findings provide a valuable reference for future investigations into the biological functions of the WRKY gene family in L. angustifolia.

Multi-Locus Sequence Typing-Based Genetic Analysis, Antifungal Resistance, and Clinical Prognosis of Cryptococcus neoformans Infections in HIV-Infected Patients in Northern China.

This study aimed to investigate the molecular epidemiological characteristics and drug sensitivity of Cryptococcus from HIV-infected patients and their relationship with patients' prognosis. Seventy-six strains were collected and identified to the species level by matrix-assisted laser desorption ionization-time of flight mass spectrometry, confirmed by internal transcribed spacer sequencing. Multi-locus sequence typing was used for the typing of Cryptococcus, and its antifungal susceptibility was tested using FUNGUS 3. The clinical outcomes of the patients were reviewed at 3-, 6-, 9-, and 12-month follow-ups. All strains were Cryptococcus neoformans var. grubii classified into seven sequence types (STs) dominated by ST5, ST31, and a new ST702 strain. The 6- and 9-month survival rates were highest for patients infected with ST31, ST32, and ST174. The antifungal resistant rates were 13.2%, 2.6%, and 1.4% for fluconazole, amphotericin B, and 5-fluorocytosine. Except itraconazole, the minimum inhibitory concentration (MIC) values and wild type (WT)/non-wild type (NWT) of Cryptococcus for antifungal drugs were not related to the clinical prognosis of HIV-infected patients with cryptococcal infection. ST5 was the main ST type, and the new ST702 type was found in a patient who died in a short period of time. Cryptococcus neoformans var. grubii had a relatively high antifungal drug resistance rate to fluconazole. The WT strain accounted for the highest proportions for 5-fluorocytosine, amphotericin B, fluconazole, voriconazole, and itraconazole. The MIC values of Cryptococcus for first-line antifungal drugs showed no relationship with clinical prognosis, implying that MIC values cannot be used to predict the clinical outcome of these patients.

A Dual‐Focus Workflow for Simultaneously Engineering High Activity and Thermal Stability in Methyl Parathion Hydrolase

AbstractIndustrial fermentation applications typically require enzymes that exhibit high stability and activity at high temperatures. However, efforts to simultaneously improve these properties are usually limited by a trade‐off between stability and activity. This report describes a computational strategy to enhance both activity and thermal stability of the mesophilic organophosphate‐degrading enzyme, methyl parathion hydrolase (MPH). To predict hotspot mutation sites, we assembled a library of features associated with the target properties for each residue and then prioritized candidate sites by hierarchical clustering. Subsequent in silico screening with multiple algorithms to simulate selective pressures yielded a subset of 23 candidate mutations. Iterative parallel screening of mutations that improved thermal stability and activity yielded, MPHase‐m5b, which exhibited 13.3 °C higher Tm and 4.2 times higher catalytic activity than wild‐type (WT) MPH over a wide temperature range. Systematic analysis of crystal structures, molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations revealed a wider entrance to the active site that increased substrate access with an extensive network of interactions outside the active site that reinforced αβ/βα sandwich architecture to improve thermal stability. This study thus provides an advanced, rational design framework to improve efficiency in engineering highly active, thermostable biocatalysts for industrial applications.

Shift in Demographic Involvement and Clinical Characteristics of COVID-19 From Wild-Type SARS-CoV-2 to the Delta Variant in the Indian Population: In Silico Analysis.

The Delta variant (B.1.617.2) was considered the most dangerous SARS-CoV-2 strain; however, in-depth studies on its impact based on demographic and clinical characteristics of COVID-19 are scarce. We aimed to investigate the shift in demographic and clinical characteristics of the COVID-19 pandemic with the emergence of the SARS-CoV-2 Delta variant compared with the wild-type (WT) strain (B.1). A cross-sectional study of COVID-19 cases in the Indian population caused by the WT strain (B.1) and Delta variant of SARS-CoV-2 was performed. The viral genomic sequence metadata containing demographic, vaccination, and patient status details (N=9500, NDelta=6238, NWT=3262) were statistically analyzed. With the Delta variant, in comparison with the WT strain, a higher proportion of young individuals (<20 years) were infected (0-9 years: Delta: 281/6238, 4.5% vs B.1: 75/3262, 2.3%; 10-19 years: Delta: 562/6238, 9% vs B.1: 229/3262, 7%; P<.001). The proportion of women contracting infection increased (Delta: 2557/6238, 41% vs B.1: 1174/3262, 36%; P<.001). However, it decreased for men (Delta: 3681/6238, 59% vs B.1: 2088/3262, 64%; P<.001). An increased proportion of the young population developed symptomatic illness and were hospitalized (Delta: 27/262, 10.3% vs B.1: 5/130, 3.8%; P=.02). Moreover, an increased proportion of the women (albeit not men) from the young (Delta: 37/262, 14.1% vs B.1: 4/130, 3.1%; P<.001) and adult (Delta: 197/262, 75.2% vs B.1: 72/130, 55.4%; P<.001) groups developed symptomatic illness and were hospitalized. The mean age of men and women who contracted infection (Delta: men=37.9, SD 17.2 years; women=36.6, SD 17.6 years; P<.001; B.1: men=39.6, SD 16.9 years; women=40.1, SD 17.4 years; P<.001) as well as developing symptoms or being hospitalized (Delta: men=39.6, SD 17.4 years; women=35.6, SD 16.9 years, P<.001; B.1: men=47, SD 18 years; women=49.5, SD 20.9 years, P<.001) were considerably lower with the Delta variant than the B.1 strain. The total mortality was about 1.8 times higher with the Delta variant than with the WT strain. With the Delta variant, compared with B.1, mortality decreased for men (Delta: 58/85, 68% vs B.1: 15/20, 75%; P<.001); in contrast, it increased for women (Delta: 27/85, 32% vs B.1: 5/20, 25%; P<.001). The odds of death increased with age, irrespective of sex (odds ratio 3.034, 95% CI 1.7-5.2, P<.001). Frequent postvaccination infections (24/6238) occurred with the Delta variant following complete doses. The increased involvement of young people and women, the lower mean age for illness, higher mortality, and frequent postvaccination infections were significant epidemiological concerns with the Delta variant.

Elevated plasma and CSF neurofilament light chain concentrations are stabilized in response to mutant huntingtin lowering in the brains of Huntington’s disease mice

BackgroundTherapeutic approaches aimed at lowering toxic mutant huntingtin (mHTT) levels in the brain can reverse disease phenotypes in animal models of Huntington's disease (HD) and are currently being evaluated in clinical trials. Sensitive and dynamic response biomarkers are needed to assess the efficacy of such candidate therapies. Neurofilament light chain (NfL) is a biomarker of neurodegeneration that increases in cerebrospinal fluid (CSF) and blood with progression of HD. However, it remains unknown whether NfL in biofluids could serve as a response biomarker for assessing the efficacy of disease-modifying therapies for HD.MethodsLongitudinal plasma and cross-sectional CSF samples were collected from the YAC128 transgenic mouse model of HD and wild-type (WT) littermate control mice throughout the natural history of disease. Additionally, biofluids were collected from YAC128 mice following intracerebroventricular administration of an antisense oligonucleotide (ASO) targeting the mutant HTT transgene (HTT ASO), at ages both before and after the onset of disease phenotypes. NfL concentrations in plasma and CSF were quantified using ultrasensitive single-molecule array technology.ResultsPlasma and CSF NfL concentrations were significantly elevated in YAC128 compared to WT littermate control mice from 9 months of age. Treatment of YAC128 mice with either 15 or 50 µg HTT ASO resulted in a dose-dependent, allele-selective reduction of mHTT throughout the brain at a 3-month interval, which was sustained with high-dose HTT ASO treatment for up to 6 months. Lowering of brain mHTT prior to the onset of regional brain atrophy and HD-like motor deficits in this model had minimal effect on plasma NfL at either dose, but led to a dose-dependent reduction of CSF NfL. In contrast, initiating mHTT lowering in the brain after the onset of neuropathological and behavioural phenotypes in YAC128 mice resulted in a dose-dependent stabilization of NfL increases in both plasma and CSF.ConclusionsOur data provide evidence that the response of NfL in biofluids is influenced by the magnitude of mHTT lowering in the brain and the timing of intervention, suggesting that NfL may serve as a promising exploratory response biomarker for HD.

Adaptive laboratory evolution of Lipomyces starkeyi for high production of lignin derivative alcohol and lipids with comparative untargeted metabolomics-based analysis.

Adaptive laboratory evolution (ALE) is an impactful technique for cultivating microorganisms to adapt to specific environmental circumstances or substrates through iterative growth and selection. This study utilized an adaptive laboratory evolution method on Lipomyces starkeyi for high tolerance in producing lignin derivative alcohols and lipids from syringaldehyde. Afterward, untargeted metabolomics analysis was employed to find the key metabolites that play important roles in the better performance of evolved strains compared to the wild type. Lignin, a prominent constituent of plant biomass, is a favorable source material for the manufacture of biofuel and lipids. Nevertheless, the effective transformation of chemicals produced from lignin into products with high economic worth continues to be a difficult task. In this study, we exposed L. starkeyi to a series of flask passaging experiments while applying selective pressure to facilitate its adaptation to syringaldehyde, a specific type of lignin monomeric aldehyde. Using ALE, we successfully developed a new strain, DALE-22, which can synthesize syringyl alcohol up to 18.74 mM from 22.28 mM syringaldehyde with 41.9% lipid accumulation. In addition, a comprehensive examination of untargeted metabolomics identified six specific crucial metabolites linked to the improved tolerance of the evolved strain in the utilization of syringaldehyde, including 2-aminobutyric acid, allantoin, 4-hydroxyphenethyl alcohol, 2-aminoethanol, tryptophan, and 5-aminovaleric acid. The results of our study reveal how L. starkeyi adapts to using substrates produced from lignin. These findings offer important information for developing strategies to improve the process of converting lignin into valuable products for sustainable biorefinery applications.

The roles of Growth Hormone dependent JAK-STAT5 and Lyn kinase signalling in determining lifespan and cancer incidence.

In rodents, loss of GH or its receptor is associated with extended lifespan. We aimed to determine the signalling process resulting in this longevity using GH receptor (GHR) mutant mice with key signalling pathways deleted and correlate this with cancer incidence and expression of genes associated with longevity. GHR uses both canonical JAK2-STAT signalling as well as signalling via LYN-ERK1/2 pathway. We utilised C57BL/6 mice with loss of key receptor tyrosines and truncation resulting in (1) loss of most STAT5 response to GH or (2) total inability to generate STAT5 to GH or (3) loss of Box1 to prevent activation of JAK2 but not LYN kinase or (4) total knockout of the receptor. For each mutant we analysed lifespan, histopathology to determine likely cause of death, and hepatic gene and protein expression. The extended lifespan is evident in the Box1 mutant males (retains Lyn activation) have median lifespan of 1016 days compared to 890 days for the Ghr-/- males. In the females, GhrBox1-/- mice have a median lifespan of 970 days compared to 911 days for the knockout females. Sexually dimorphic GHR-STAT5 is repressive for longevity, since its removal results in a median lifespan of 1003 days in females compared to 734 days for wild type females. Numerous transcripts related to insulin sensitivity, oxidative stress response and mitochondrial function are regulated by GHR-STAT5, however LYN responsive genes involve DNA repair, cell cycle control, and anti-inflammatory response. There appears to be a yin-yang relationship between JAK2 and LYN that determines lifespan.

Chlorophyll to zeaxanthin energy transfer in nonphotochemical quenching: An exciton annihilation-free transient absorption study

Zeaxanthin (Zea) is a key component in the energy-dependent, rapidly reversible, nonphotochemical quenching process (qE) that regulates photosynthetic light harvesting. Previous transient absorption (TA) studies suggested that Zea can participate in direct quenching via chlorophyll (Chl) to Zea energy transfer. However, the contamination of intrinsic exciton-exciton annihilation (EEA) makes the assignment of TA signal ambiguous. In this study, we present EEA-free TA data using Nicotiana benthamiana thylakoid membranes, including the wild type and three NPQ mutants (npq1, npq4, and lut2) generated by CRISPR/Cas9 mutagenesis. The results show a strong correlation between excitation energy transfer from excited Chl Qy to Zea S1 and the xanthophyll cycle during qE activation. Notably, a Lut S1 signal is absent in the npq1 thylakoids which lack zeaxanthin. Additionally, the fifth-order response analysis shows a reduction in the exciton diffusion length (LD) from 62 ± 6 nm to 43 ± 3 nm under high light illumination, consistent with the reduced range of exciton motion being a key aspect of plants' response to excess light.

Proteomic analysis reveals molecular changes following genetic engineering in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is gaining recognition as a promising expression system for the production of recombinant proteins. However, its performance as a cellular biofactory remains suboptimal, especially with respect to consistent expression of heterologous genes. Gene silencing mechanisms, position effect, and low nuclear transgene expression are major drawbacks for recombinant protein production in this model system. To unveil the molecular changes following transgene insertion, retention, and expression in this species, we genetically engineered C. reinhardtii wild type strain 137c (strain cc-125 mt+) to express the fluorescent protein mVenus and subsequently analysed its intracellular proteome. The obtained transgenic cell lines showed differences in abundance in more than 400 proteins, with multiple pathways altered post-transformation. Proteins involved in chromatin remodelling, translation initiation and elongation, and protein quality control and transport were found in lower abundance. On the other hand, ribosomal proteins showed higher abundance, a signal of ribosomal stress response. These results provide new insights into the modifications of C. reinhardtii proteome after transformation, highlighting possible pathways involved in gene silencing. Moreover, this study identifies multiple protein targets for future genetic engineering approaches to improve the prospective use of C. reinhardtii as cell biofactory for industrial applications.

A Novel Homozygous Variant in CPLANE1 Gene in a Patient with Developmental Deficits

Background: Oral-facial-digital syndrome (OFDS) type 6 is a rare subtype of Joubert syndrome characterized by orofacial anomalies and polydactyly with neurological features of Joubert syndrome. This rare syndrome is divided into thirteen subtypes, all of which demonstrate autosomal recessive inheritance, except for OFDS type 1 which demonstrates X-linked dominant inheritance. Case Presentation: A 19-year-old man with mild developmental delay was brought to a rural community clinic, as he had become irritable and angry, in the recent past. There was no history of prior medical conditions. In view of orofacial anomalies, and developmental deficits, a genetic analysis was requested. Karyotype analysis revealed a normal male karyotype (46,XY) in all 30 metaphase spreads analyzed. No numerical or structural chromosomal abnormalities were observed. Clinical exome sequencing and chromosomal microarray detected a variant of uncertain significance in exon 5 of CPLANE1 gene c.365T&amp;gt;G (p.Val122Gly) leading to substitution of Glycine for Valine. This was confirmed by Sanger sequencing. Parents were heterozygous, and the unaffected sibling was homozygous for the wild-type allele. This variant has not been reported earlier in the mutation databases or gnomAD. Runs of homozygosity (ROH) analysis showed a 3.2 Mb ROH around the CPLANE1 gene in the proband, which was absent in both parents and the unaffected sibling. Conclusion: We find a novel homozygous mutation in the CPLANE1 gene in a patient of non-consanguineous parentage with atypical orofacial features. This suggests that potentially deleterious, rare variants may occur in the heterozygous state in the population. Hence, sequencing of population samples might help understand the genetic epidemiology of rare syndromes.

The Role of Complement C1qa in Experimental Intracerebral Hemorrhage.

Evidence indicates that the complement system is activated and plays a role in brain injury after intracerebral hemorrhage (ICH). Most studies have focused on the role of C3, C5 and the membrane attack complex. The purpose of this study was to investigate the potential impact of complement C1q, a key upstream component of the classical pathway, on ICH-induced brain injury. Wild-type (WT) and C1qa knock out (KO) mice were compared using an autologous blood injection ICH model. Magnetic resonance imaging (MRI) was performed on days 1, 3 and 7 and brains harvested on days 3 and 7 for immunohistochemistry to examine brain injury mechanisms. WT and C1qa KO mice also received an intracerebral injection of thrombin, a key factor in ICH-induced brain injury. Following MRI scans, brains were harvested for immunohistochemistry on day 1. In comparison to WT mice, C1qa KO mice had reduced hematoma erythrolysis and neutrophil infiltration after ICH. However, they also had delayed hematoma clearance, which was associated with reduced induction of phagocytic multinuclear giant cells, and increased perihematomal neuronal damage. After thrombin injection, C1qa KO mice had smaller lesion volumes, less neuronal loss, reduced neutrophil infiltration, and less BBB damage. C1qa knockout has beneficial and detrimental effects on ICH-induced brain injury mechanisms, but a consistent beneficial effect after thrombin injection. Strategies to balance the roles of C1q after ICH may represent a promising therapeutic direction.