Early Gene Research Articles

Serum IgG antibodies to HPV6 L1, E2, E4, E6, and E7 proteins among children prospectively followed-up for three years.

Current knowledge implicates that human papillomavirus (HPV) infection can be acquired at early age. However, the role of HPV-specific passive immunization from mother to neonate is nearly unexplored, especially against the HPV early proteins. We analyzed IgG antibodies against HPV6 early (E2, E4, E6, E7) and late (L1) proteins in children prospectively followed-up for three years. A total of 272 children and their mothers from the Finnish Family HPV Study were included in these analyses. Serum samples were obtained from pregnant mothers at their third trimester and from newborn/infants at 1-, 2-, 6-, 12-, 24-, and 36-month visits after birth. Antibodies to the early and late proteins were analyzed by multiplex serology based on glutathione S-transferase fusion protein capture to fluorescent beads. Maternal antibodies to all tested HPV6 proteins were transferred to neonates, concordance between maternal and neonates' antibody levels being highly significant (p<0.001). Seropositivity of HPV6 L1 in the neonates declined during the first six months of life, whereas changes in the E-protein antibodies were less obvious. After the maternal antibodies have vanished, seroconversion to HPV6 L1 at 12 months (median) and to the HPV6 E-proteins between 23-35 months was observed. IgG antibodies against HPV6 E- and L-proteins are transferred from mothers to their children. Seroconversion against HPV6 L1, E2, E4, E6, and E7 does occur in early childhood, as a sign of acquired HPV6 infection by vertical or horizontal transmission starting at 12 months of age.

Behavioral transcriptomic effects of triploidy and probiotic therapy (Bifidobacterium, Lactobacillus, and Lactococcus mixture) on juvenile Chinook salmon (Oncorhynchus tshawytscha).

Aquaculturists use polyploid fish to maximize production albeit with some unintended consequences including compromised behaviors and physiological function. Given benefits of probiotic therapies (e.g., improved immune response, growth, and metabolism), we explored probiotic supplementation (mixture of Bifidobacterium, Lactobacillus, and Lactococcus), to overcome drawbacks. We first examined fish gut bacterial community composition using 16S metabarcoding (via principal coordinate analyses and PERMANOVA) and determined probiotics significantly impacted gut bacteria composition (p = 0.001). Secondly, we examined how a genomic disruptor (triploidy) and diet supplements (probiotics) impact gene transcription and behavioral profiles of hatchery-reared Chinook salmon (Oncorhynchus tshawytscha). Juveniles from four treatment groups (diploid-regular feed, diploid-probiotic feed, triploid-regular feed, and triploid-probiotic feed; n = 360) underwent behavioral assays to test activity, exploration, neophobia, predator evasion, aggression/sociality, behavioral sensitivity, and flexibility. In these fish, transcriptional profiles for genes associated with neural functions (neurogenesis/synaptic plasticity) and biomarkers for stress response and development (growth/appetite) were (i) examined across treatments and (ii) used to describe behavioral phenotypes via principal component analyses and general linear mixed models. Triploids exhibited a more active behavioral profile (p = 0.002), and those on a regular diet had greater Neuropeptide Y transcription (p = 0.02). A growth gene (early growth response protein 1, p = 0.02) and long-term neural development genes (neurogenic differentiation factor, p = 0.003 and synaptysomal-associated protein 25-a, p = 0.005) impacted activity and reactionary profiles, respectively. Overall, our probiotic treatment did not compensate for triploidy. Our research highlights novel applications of behavioral transcriptomics for identifying candidate genes and dynamic, mechanistic associations with complex behavioral repertoires.

Exploring ketamine’s reinforcement, cue-induced reinstatement, and nucleus accumbens cFos activation in male and female long evans rats

Ketamine (KET), a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, has rapid onset of antidepressant effects in Treatment-Resistant Depression patients and repeated infusions are required to sustain its antidepressant properties. However, KET is an addictive drug, and so more preclinical and clinical research is needed to assess the safety of recurring treatments in both sexes. Thus, the aim of this study was to investigate the reinforcing properties of various doses of KET (0-, 0.125-, 0.25-, 0.5 mg/kg/infusion) and assess KET’s cue-induced reinstatement and neuronal activation in both sexes of Long Evans rats. Neuronal activation was assessed using the protein expression of the immediate early gene cFos in the nucleus accumbens (Nac), an important brain area implicated in reward, reinforcement and reinstatement to most drug-related cues. Our findings show that KET has reinforcing effects in both male and female rats, albeit exclusively at the highest two doses (0.25 and 0.5 mg/kg/infusion). Furthermore, we noted sex differences, particularly at the highest dose of ketamine, with female rats displaying a higher rate of self-administration. Interestingly, all groups that self-administered KET reinstated to drug-cues. Following drug cue-induced reinstatement test in rats exposed to KET (0.25 mg/kg/infusion) or saline, there was higher cFos protein expression in KET-treated animals compared to saline controls, and higher cFos expression in the core compared to the shell subregions of the Nac. As for reinstatement, there were no notable sex differences reported for cFos expression in the Nac. These findings reveal some sex and dose dependent effects in KET’s reinforcing properties and that KET at all doses induced similar reinstatement in both sexes. This study also demonstrated that cues associated with ketamine induce comparable neuronal activation in the Nac of both male and female rats. This work warrants further research into the potential addictive properties of KET, especially when administered at lower doses which are now being used in the clinic for treating various psychopathologies.

#1293 Endothelial calcification induced by high-phosphate is characterized by mesenchymal and osteoblastic transdifferentiation

Abstract Background and Aims Chronic kidney disease (CKD) is a prevalent condition that affects millions of people. Patients with CKD have an increased cardiovascular risk, mostly due to vascular calcification (VC). By now, it's quite clear how, in vitro and in vivo, vascular smooth muscle cells (VSMCs) actively participate to VC. In contrast to the wide studies on VSMCs, the role of endothelial cells (ECs) has been poorly investigated. It's known that ECs have a dynamic nature and following particular stimuli they can trans-differentiate and acquire several different phenotypes through the endothelial-to-mesenchymal transition (EndMT). Methods Human aortic ECs (HAECs) were challenged, in an in vitro calcification model, with 2, 2.5, 3, 3.5 mM Pi for up to 7 days. Early morphological changes, gene expression and cell migration were evaluated. Afterwards calcium deposition, genes and protein expression and ultrastructural changes were analyzed. Results High-Pi, at a concentration of 3 mM, is able to induce EC calcium deposition. Calcification is dependent on calcium influx without influencing Pit-1 expression. During the first days of high-Pi treatment ECs change their morphology from cobblestone to spindle shape acquiring motility, as demonstrated by the 24% increased closure in the scratch wound test. EndMT master genes such as SNAIL and TWIST resulted up-regulated at day 3 together with an increased protein expression of mesenchymal markers such as N-Cadherin, α-SMA, SM22α, FSP-1 and Fibronectin. Starting from day 5 high-Pi induces osteoblastic differentiation with RUNX2 and BMP2 mRNA up-regulation up to day 7, osteopontin increased protein expression and positive Alcian Blu staining. In calcified ECs mesenchymal and endothelial markers, such as α-SMA, SM22α, FSP-1 and Fibronectin or vWF progressively decrease. Finally, uremic serum is able to exacerbate in vitro calcification, compared to healthy serum characterized by an increase in both mesenchymal and osteoblastic mastergene such as TWIST and RUNX2. Conclusions Our data demonstrate that in vitro high-Pi induces the EndMT process and similar osteoblastic trans-differentiation suggesting a potential role of endothelium in VC. The relevancy of endothelial calcification is suggested by the calcification exacerbation induced by uremic compared to healthy sera.

#2106 Higher daily protein intake can reduce the risk of infections in kidney transplant recipients during the early post-transplant period

Abstract Background and Aims Kidney transplantation (KT) is considered the most effective treatment for end-stage renal disease (ESRD), and it significantly contributes to enhancing the quality of life and extending the survival of patients with ESRD. After kidney transplantation, the early and heavy use of immunosuppressants and glucocorticoids can affect the recipients' immune status to varying degrees, increasing the risk of postoperative infections caused by a wide range of pathogens. Thus, the prevention and management of infectious diseases are major factors contributing to improved outcomes in kidney transplantation. Protein-energy wasting (PEW) is commonly observed in uremic patients, and it may persist even after they have undergone kidney transplantation (KT). And PEW is a significant factor in the progression of chronic kidney disease (CKD). Providing appropriate dietary guidance and intervention is essential for enhancing the immune status of recipients, promoting a positive nitrogen balance in the body, minimizing infections, and safeguarding graft function. This study aimed to investigate the relationship between daily protein intake (DPI) and postoperative infections at 3 months post-transplant, which may establish a foundation for the early postoperative protein intake regimen, reducing the incidence of postoperative infections, and improving the early prognosis of kidney transplantation. Method This is a retrospective observational study. 176 kidney transplant recipients (KTRs) who underwent kidney transplant in West China Hospital from December 2021 to June 2022 were included and followed up regularly. 24-hour urine urea data were collected at months 1 and 3 post-transplant to calculate their DPI. These KTRs were further divided into two groups according to the recommended DPI for KTRs by the 2021 Practice Guidelines for the Nutritional Management of Chronic Kidney Disease, namely high intake group (Group 1, with DPI ≥ 1.4 g/kg·d, n = 66) and low intake group (Group 2, with DPI &amp;lt; 1.4 g/kg·d, n = 110). Occurrence of hospitalization and postoperative infections within 3 months were recorded. Multiple logistic regression was utilized to analyze the association between DPI and early postoperative infections at 3 months post-transplant. Results Mean DPI levels at month 1 post-transplant were 1.58 ± 0.22 g/kg·d in Group 1 and 1.02 ± 0.31 g/kg·d in Group 2, respectively (P &amp;lt; 0.05), this huge difference in DPI between the two groups continued to month 3. There was no significant difference in age, gender, pre-transplant body mass index (BMI), dialysis type and time, donor type, or the ratio of delayed graft function (DGF) between the two groups. There was a statistically significant difference in the percentage of hospitalization between the two groups during the postoperative period of 3 months (P = 0.027). Pneumonia was the most common reason of hospitalization after transplantation, followed by urinary tract infection. The results of multiple logistic regression analysis showed that higher DPI in the early post-transplant period was a protective factor for infection after kidney transplantation. Conclusion In the early post-transplant period, consuming higher levels of dietary protein may reduce the risk of infections and complications, and help preserve the function of the transplanted kidney.

#2736 Higher daily protein intake can reduce the risk of infections in kidney transplant recipients during the early post-transplant period

Abstract Background and Aims Kidney transplantation (KT) is considered the most effective treatment for end-stage renal disease (ESRD), and it significantly contributes to enhancing the quality of life and extending the survival of patients with ESRD. After kidney transplantation, the early and heavy use of immunosuppressants and glucocorticoids can affect the recipients' immune status to varying degrees, increasing the risk of postoperative infections caused by a wide range of pathogens. Thus, the prevention and management of infectious diseases are major factors contributing to improved outcomes in kidney transplantation. Protein-energy wasting (PEW) is commonly observed in uremic patients, and it may persist even after they have undergone kidney transplantation (KT). And PEW is a significant factor in the progression of chronic kidney disease (CKD). Providing appropriate dietary guidance and intervention is essential for enhancing the immune status of recipients, promoting a positive nitrogen balance in the body, minimizing infections, and safeguarding graft function. This study aimed to investigate the relationship between daily protein intake (DPI) and postoperative infections at 3 months post-transplant, which may establish a foundation for the early postoperative protein intake regimen, reducing the incidence of postoperative infections, and improving the early prognosis of kidney transplantation. Method This is a retrospective observational study. 176 kidney transplant recipients (KTRs) who underwent kidney transplant in West China Hospital from December 2021 to June 2022 were included and followed up regularly. 24-hour urine urea data were collected at month 3 post-transplant to calculate their DPI. These KTRs were further divided into two groups according to the recommended DPI for KTRs by the 2021 Practice Guidelines for the Nutritional Management of Chronic Kidney Disease, namely high intake group (Group 1, with DPI ≥ 1.4 g/kg·d, n = 66) and low intake group (Group 2, with DPI &amp;lt; 1.4 g/kg·d, n = 110). Occurrence of postoperative infections events within 3 months were recorded. Multiple logistic regression was utilized to analyze the association between DPI and early postoperative infections at 3 months post-transplant. Results Mean DPI levels at month 3 post-transplant were 1.75 ± 0.56 g/kg·d in Group 1 and 0.98 ± 0.24 g/kg·d in Group 2, respectively. There was no significant difference in age, gender, pre-transplant body mass index (BMI), dialysis type and time, donor type, or the ratio of delayed graft function (DGF) between the two groups. There was a statistically significant difference in the percentage of hospitalization for infections between the two groups during the postoperative period of 3 months (P = 0.027). Pneumonia was the most common reason of rehospitalization after transplantation, followed by urinary tract infection. The results of multiple logistic regression analysis showed that higher DPI in the early post-transplant period was a protective factor for infection after kidney transplantation. Conclusion In the early post-transplant period, consuming higher levels of dietary protein can reduce the risk of infections and complications, and help preserve the function of the transplanted kidney.

CRISPR-Cas Genome Editing in Ex Vivo Human Lungs to Rewire the Translational Path of Genome-Targeting Therapeutics.

The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both invivo and ex vivo organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients. However, assessing genome-targeting interventions in a clinically relevant manner prior to clinical trials remains a major challenge owing to the limited modalities available. This study introduces a novel platform for testing genome editing in human lungs ex vivo, effectively simulating preimplantation genetic engineering of donor organs. We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene interleukin 10 (IL-10). We combined this approach with adenoviral vector-mediated IL-10 delivery to create favorable kinetics for early (immediate postimplantation) graft immunomodulation. Using ex vivo organ machine perfusion and precision-cut tissue slice technology, we demonstrated the feasibility of evaluating CRISPR genome editing in human lungs. To overcome the assessment limitations in ex vivo perfused human organs, we conducted an invivo rodent study and demonstrated both early gene induction and sustained editing of the lung. Collectively, our findings lay the groundwork for a first-in-human-organ study to overcome the current translational barriers of genome-targeting therapeutics.

Aberrant RNA polymerase initiation and processivity on the genome of a herpes simplex virus 1 mutant lacking ICP27.

Within the first 15 minutes of infection, herpes simplex virus 1 immediate early proteins repurpose cellular RNA polymerase (Pol II) for viral transcription. An important role of the viral-infected cell protein 27 (ICP27) is to facilitate viral pre-mRNA processing and export viral mRNA to the cytoplasm. Here, we use precision nuclear run-on followed by deep sequencing (PRO-seq) to characterize transcription of a viral ICP27 null mutant. At 1.5 and 3 hours post infection (hpi), we observed increased total levels of Pol II on the mutant viral genome and accumulation of Pol II downstream of poly A sites indicating increased levels of initiation and processivity. By 6 hpi, Pol II accumulation on specific mutant viral genes was higher than that on wild-type virus either at or upstream of poly A signals, depending on the gene. The PRO-seq profile of the ICP27 mutant on late genes at 6 hpi was similar but not identical to that caused by treatment with flavopiridol, a known inhibitor of RNA processivity. This pattern was different from PRO-seq profiles of other α gene mutants and upon inhibition of viral DNA replication with PAA. Together, these results indicate that ICP27 contributes to the repression of aberrant viral transcription at 1.5 and 3 hpi by inhibiting initiation and decreasing RNA processivity. However, ICP27 is needed to enhance processivity on most late genes by 6 hpi in a mechanism distinguishable from its role in viral DNA replication.IMPORTANCEWe developed and validated the use of a processivity index for precision nuclear run-on followed by deep sequencing data. The processivity index calculations confirm infected cell protein 27 (ICP27) induces downstream of transcription termination on certain host genes. The processivity indices and whole gene probe data implicate ICP27 in transient immediate early gene-mediated repression, a process that also requires ICP4, ICP22, and ICP0. The data indicate that ICP27 directly or indirectly regulates RNA polymerase (Pol II) initiation and processivity on specific genes at specific times post infection. These observations support specific and varied roles for ICP27 in regulating Pol II activity on viral genes in addition to its known roles in post transcriptional mRNA processing and export.

The impact of pubertal stress and adult hormone exposure on the transcriptome of the developing hypothalamus

Why individuals suffer negative consequences following stress is a complex phenomenon that is dictated by individual factors, the timing of stress within the lifespan, and when in the lifespan the consequences are measured. Women who undergo adverse childhood experiences are at risk for lasting biological consequences, including affective and stress dysregulation. We have shown that pubertal adversity is associated with a blunted hypothalamic-pituitary-adrenal axis glucocorticoid response in peripartum humans and mice. In mice, our prior examination of the paraventricular nucleus (PVN) of the hypothalamus showed that pubertal stress led to an upregulation of baseline mRNA expression of six immediate early genes (IEGs) in the PVN of adult, pregnant mice. Separately, we showed that the pregnancy-associated hormone allopregnanolone is necessary and sufficient to produce the blunted stress response phenotype in pubertally stressed mice. In the current study, we further examined a potential mechanistic role for the IEGs in the PVN. We found that in pubertally stressed adult female, but not male, mice, intra-PVN allopregnanolone was sufficient to recapitulate the baseline IEG mRNA expression profile previously observed in pubertally stressed, pregnant mice. We also examined baseline IEG mRNA expression during adolescence, where we found that IEGs have developmental trajectories that showed sex-specific disruption by pubertal stress. Altogether, these data establish that IEGs may act as a key molecular switch involved in increased vulnerability to negative outcomes in adult, pubertally stressed animals. How the factors that produce vulnerability combine throughout the lifespan is key to our understanding of the etiology of stress-related disorders.

A helping HAND: therapeutic potential of MAGL inhibition against HIV-1-associated neuroinflammation.

Human immunodeficiency virus (HIV) affects nearly 40 million people globally, with roughly 80% of all people living with HIV receiving antiretroviral therapy. Antiretroviral treatment suppresses viral load in peripheral tissues but does not effectively penetrate the blood-brain barrier. Thus, viral reservoirs persist in the central nervous system and continue to produce low levels of inflammatory factors and early viral proteins, including the transactivator of transcription (Tat). HIV Tat is known to contribute to chronic neuroinflammation and synaptodendritic damage, which is associated with the development of cognitive, motor, and/or mood problems, collectively known as HIV-associated neurocognitive disorders (HAND). Cannabinoid anti-inflammatory effects are well documented, but therapeutic utility of cannabis remains limited due to its psychotropic effects, including alterations within brain regions encoding reward processing and motivation, such as the nucleus accumbens. Alternatively, inhibiting monoacylglycerol lipase (MAGL) has demonstrated therapeutic potential through interactions with the endocannabinoid system. The present study utilized a reward-related operant behavioral task to quantify motivated behavior in female Tat transgenic mice treated with vehicle or MAGL inhibitor MJN110 (1 mg/kg). Brain tissue was collected to assess dendritic injury and neuroinflammatory profiles, including dendritic microtubule-associated protein (MAP2ab) intensity, microglia density, microglia morphology, astrocyte density, astrocytic interleukin-1ß (IL-1ß) colocalization, and various lipid mediators. No significant behavioral differences were observed; however, MJN110 protected against Tat-induced dendritic injury by significantly upregulating MAP2ab intensity in the nucleus accumbens and in the infralimbic cortex of Tat(+) mice. No or only minor effects were noted for Iba-1+ microglia density and/or microglia morphology. Further, Tat increased GFAP+ astrocyte density in the infralimbic cortex and GFAP+ astrocytic IL-1ß colocalization in the nucleus accumbens, with MJN110 significantly reducing these measures in Tat(+) subjects. Lastly, selected HETE-related inflammatory lipid mediators in the striatum were downregulated by chronic MJN110 treatment. These findings demonstrate anti-inflammatory and neuroprotective properties of MJN110 without cannabimimetic behavioral effects and suggest a promising alternative to cannabis for managing neuroinflammation.

Reversibly Reactive Affinity Selection-Mass Spectrometry Enables Identification of Covalent Peptide Binders.

Covalent peptide binders have found applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and tunable affinity selection platform to enrich covalent reactive peptide binders from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform termed ReAct-ASMS enabled by tandem high-resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfide-containing peptides to build reversible peptide-protein conjugates that can enrich for covalent variants, which can be sequenced by MS/MS after reduction. Using this platform, we identified covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pin1). The resulting peptide binders efficiently and selectively cross-link Cys58 of E6 at 37 °C and Cys113 of Pin1 at room temperature, respectively. ReAct-ASMS enables the identification of highly selective covalent peptide binders for diverse molecular targets, introducing an applicable platform to assist preclinical therapeutic development pipelines.

WSSV early protein WSSV004 enhances viral replication by suppressing LDH activity

White spot syndrome virus (WSSV) is known to upregulate glycolysis to supply biomolecules and energy for the virus's replication. At the viral genome replication stage, lactate dehydrogenase (LDH), a glycolytic enzyme, shows increased activity without any increase in expression. In the present study, yeast 2-hybrid screening was used to identify WSSV proteins that interacted with LvLDH isoform 1 and 2, and these included the WSSV early protein WSSV004. The interaction between WSSV004 and LvLDH1/2 was confirmed by co-immunoprecipitation. Immunofluorescence showed that WSSV004 co-localized with LvLDH1/2 in the cytoplasm. dsRNA silencing experiments showed that WSSV004 was crucial for WSSV replication. However, although WSSV004 silencing led to the suppression of total LvLDH gene expression during the viral late stage, there was nevertheless a significant increase in LvLDH activity at this time. We also used affinity purification-mass spectrometry to identify cellular proteins that interact with WSSV004, and found a total of 108 host proteins and 3 WSSV proteins with which it potentially interacts. Bioinformatics analysis revealed that WSSV004 and its interacting proteins might be responsible for various biological pathways during infection, including vesicular transport machinery and RNA-related functions. Collectively, our study suggests that WSSV004 serves as a multifunctional modulator to facilitate WSSV replication.

The nucleic acid binding protein SFPQ represses EBV lytic reactivation by promoting histone H1 expression.

Epstein-Barr virus (EBV) uses a biphasic lifecycle of latency and lytic reactivation to infect >95% of adults worldwide. Despite its central role in EBV persistence and oncogenesis, much remains unknown about how EBV latency is maintained. We used a human genome-wide CRISPR/Cas9 screen to identify that the nuclear protein SFPQ was critical for latency. SFPQ supported expression of linker histone H1, which stabilizes nucleosomes and regulates nuclear architecture, but has not been previously implicated in EBV gene regulation. H1 occupied latent EBV genomes, including the immediate early gene BZLF1 promoter. Upon reactivation, SFPQ was sequestered into sub-nuclear puncta, and EBV genomic H1 occupancy diminished. Enforced H1 expression blocked EBV reactivation upon SFPQ knockout, confirming it as necessary downstream of SFPQ. SFPQ knockout triggered reactivation of EBV in B and epithelial cells, as well as of Kaposi's sarcoma-associated herpesvirusin B cells, suggesting a conserved gamma-herpesvirus role. These findings highlight SFPQ as a major regulator of H1 expression and EBV latency.

Phage protein Gp11 blocks Staphylococcus aureus cell division by inhibiting peptidoglycan biosynthesis.

Understanding the interplay between phages and their hosts is important for the development of novel therapies against pathogenic bacteria. Although phages have been used to control methicillin-resistant Staphylococcus aureus infections, our knowledge related to the processes in the early stages of phage infection is still limited. Owing to the fact that most of the phage early proteins have been classified as hypothetical proteins with uncertain functions, we screened phage early-gene products that inhibit cell growth in S. aureus, and one protein, Gp11, selectively targets essential host genes to block the synthesis of the peptidoglycan component lipid II, ultimately leading to cell growth arrest in S. aureus. Our study provides a novel insight into the strategy by which Gp11 blocks essential host cellular metabolism to influence phage-host interaction. Importantly, dissecting the interactions between phages and host cells will contribute to the development of new and effective therapies to treat bacterial infections.

Short tandem repeat expansions in cortical layer-specific genes implicate in phenotypic severity and adaptability of autism spectrum disorder.

Short tandem repeats (STRs) are repetitive DNA sequences and highly mutable in various human disorders. While the involvement of STRs in various genetic disorders has been extensively studied, their role in autism spectrum disorder (ASD) remains largely unexplored. In this study, we aimed to investigate genetic association of STR expansions with ASD using whole genome sequencing (WGS) and identify risk loci associated with ASD phenotypes. We analyzed WGS data of 634 ASD families and performed genome-wide evaluation for 12,929 STR loci. We found rare STR expansions that exceeded normal repeat lengths in autism cases compared to unaffected controls. By integrating single cell RNA and ATAC sequencing datasets of human postmortem brains, we prioritized STR loci in genes specifically expressed in cortical development stages. A deep learning method was used to predict functionality of ASD-associated STR loci. In ASD cases, rare STR expansions predominantly occurred in early cortical layer-specific genes involved in neurodevelopment, highlighting the cellular specificity of STR-associated genes in ASD risk. Leveraging deep learning prediction models, we demonstrated that these STR expansions disrupted the regulatory activity of enhancers and promoters, suggesting a potential mechanism through which they contribute to ASD pathogenesis. We found that individuals with ASD-associated STR expansions exhibited more severe ASD phenotypes and diminished adaptability compared to non-carriers. Short tandem repeat expansions in cortical layer-specific genes are associated with ASD and could potentially be a risk genetic factor for ASD. Our study is the first to show evidence of STR expansion associated with ASD in an under-investigated population.

Two sequential gene expression programs bridged by cell division support long-distance collective cell migration.

The precise assembly of tissues and organs relies on spatiotemporal regulation of gene expression to coordinate the collective behavior of cells. In Drosophila embryos, the midgut musculature is formed through collective migration of caudal visceral mesoderm (CVM) cells, but how gene expression changes as cells migrate is not well understood. Here, we have focused on ten genes expressed in the CVM and the cis-regulatory sequences controlling their expression. Although some genes are continuously expressed, others are expressed only early or late during migration. Late expression relates to cell cycle progression, as driving string/Cdc25 causes earlier division of CVM cells and accelerates the transition to late gene expression. In particular, we found that the cell cycle effector transcription factor E2F1 is a required input for the late gene CG5080. Furthermore, whereas late genes are broadly expressed in all CVM cells, early gene transcripts are polarized to the anterior or posterior ends of the migrating collective. We show this polarization requires transcription factors Snail, Zfh1 and Dorsocross. Collectively, these results identify two sequential gene expression programs bridged by cell division that support long-distance directional migration of CVM cells.

USP20 deubiquitinates and stabilizes the reticulophagy receptor RETREG1/FAM134B to drive reticulophagy

ABSTRACT The endoplasmic reticulum (ER) serves as a hub for various cellular processes, and maintaining ER homeostasis is essential for cell function. Reticulophagy is a selective process that removes impaired ER subdomains through autophagy-mediatedlysosomal degradation. While the involvement of ubiquitination in autophagy regulation is well-established, its role in reticulophagy remains unclear. In this study, we screened deubiquitinating enzymes (DUBs) involved in reticulophagy and identified USP20 (ubiquitin specific peptidase 20) as a key regulator of reticulophagy under starvation conditions. USP20 specifically cleaves K48- and K63-linked ubiquitin chains on the reticulophagy receptor RETREG1/FAM134B (reticulophagy regulator 1), thereby stabilizing the substrate and promoting reticulophagy. Remarkably, despite lacking a transmembrane domain, USP20 is recruited to the ER through its interaction with VAPs (VAMP associated proteins). VAPs facilitate the recruitment of early autophagy proteins, including WIPI2 (WD repeat domain, phosphoinositide interacting 2), to specific ER subdomains, where USP20 and RETREG1 are enriched. The recruitment of WIPI2 and other proteins in this process plays a crucial role in facilitating RETREG1-mediated reticulophagy in response to nutrient deprivation. These findings highlight the critical role of USP20 in maintaining ER homeostasis by deubiquitinating and stabilizing RETREG1 at distinct ER subdomains, where USP20 further recruits VAPs and promotes efficient reticulophagy. Abbreviations: ACTB actin beta; ADRB2 adrenoceptor beta 2; AMFR/gp78 autocrine motility factor receptor; ATG autophagy related; ATL3 atlastin GTPase 3; BafA1 bafilomycin A1; BECN1 beclin 1; CALCOCO1 calcium binding and coiled-coil domain 1; CCPG1 cell cycle progression 1; DAPI 4’,6-diamidino-2-phenylindole; DTT dithiothreitol; DUB deubiquitinating enzyme; EBSS Earle’s Balanced Salt Solution; FFAT two phenylalanines (FF) in an acidic tract; GABARAP GABA type A receptor-associated protein; GFP green fluorescent protein; HMGCR 3-hydroxy-3-methylglutaryl-CoA reductase; IL1B interleukin 1 beta; LIR LC3-interacting region; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; PIK3C3/Vps34 phosphatidylinositol 3-kinase catalytic subunit type 3; RB1CC1/FIP200 RB1 inducible coiled-coil 1; RETREG1/FAM134B reticulophagy regulator 1; RFP red fluorescent protein; RHD reticulon homology domain; RIPK1 receptor interacting serine/threonine kinase 1; RTN3L reticulon 3 long isoform; SEC61B SEC61 translocon subunit beta; SEC62 SEC62 homolog, preprotein translocation factor; SIM super-resolution structured illumination microscopy; SNAI2 snail family transcriptional repressor 2; SQSTM1/p62 sequestosome 1; STING1/MITA stimulator of interferon response cGAMP interactor 1; STX17 syntaxin 17; TEX264 testis expressed 264, ER-phagy receptor; TNF tumor necrosis factor; UB ubiquitin; ULK1 unc-51 like autophagy activating kinase 1; USP20 ubiquitin specific peptidase 20; USP33 ubiquitin specific peptidase 33; VAMP8 vesicle associated membrane protein 8; VAPs VAMP associated proteins; VMP1 vacuole membrane protein 1; WIPI2 WD repeat domain, phosphoinositide interacting 2; ZFYVE1/DFCP1 zinc finger FYVE-type containing 1.

A new compound, phomaherbarine A, induces cytolytic reactivation in epstein-barr virus-positive B cell lines

Epstein-Barr virus (EBV), the first virus found to induce cancer in humans, has been frequently detected in various types of B cell lymphomas. During its latent phase, EBV expresses a limited set of proteins crucial for its persistence. Induction of the lytic phase of EBV has shown promise in the treatment of EBV-associated malignancies. The present study assessed the ability of phomaherbarine A, a novel compound derived from the endophytic fungus Phoma herbarum DBE-M1, to stimulate lytic replication of EBV in B95-8 cells. Phomaherbarine A was found to efficiently initiate the expression of both early and late EBV lytic genes in B95-8 cells, with this initiation being further heightened by the addition of phorbol myristate acetate and sodium butyrate. Moreover, phomaherbarine A demonstrated notable cytotoxicity against the EBV-associated B cell lymphoma cell lines B95-8 and Raji. Mechanistically, phomaherbarine A induces apoptosis in these cells through the activation of caspase-3/7. When combined with ganciclovir, phomaherbarine A does not interfere with the reduction of viral replication by ganciclovir and sustains its apoptosis induction. In conclusion, these findings indicate that phomaherbarine A may be a promising candidate for therapeutic intervention in patients with EBV-associated B cell lymphomas.

Comparative genome analysis of three classical E. coli cloning strains designed for blue/white selection: JM83, JM109 and XL1-Blue.

The development of the Escherichia coli K-12 laboratory strains JM83, JM109 and XL1-Blue was instrumental in early gene technology. We report the comprehensive genome sequence analysis of JM83 and XL1-Blue using Illumina and Oxford Nanopore technologies and a comparison with both the wild-type sequence (MG1655) and the genome of JM109 deposited at GenBank. Our investigation provides insight into the way how the genomic background that allows blue/white colony selection-by complementing a functionally inactive ω-fragment of β-galactosidase (LacZ) with its α-peptide encoded on the cloning vector-has been implemented independently in these three strains using classical bacterial genetics. In fact, their comparative analysis reveals recurrent motifs: (i) inactivation of the native enzyme via large deletions of chromosomal regions encompassing the lac locus, or a chemically induced frameshift deletion at the beginning of the lacZ cistron, and (ii) utilization of a defective prophage (ϕ80), or an F'-plasmid, to provide the lacZ∆M15 allele encoding its ω-fragment. While the genetic manipulations of the E. coli strains involved repeated use of mobile genetic elements as well as harsh chemical or physical mutagenesis, the individual modified traits appear remarkably stable as they can be found even in distantly related laboratory strains, beyond those investigated here. Our detailed characterization at the genome sequence level not only offers clues about the mechanisms of classical gene transduction and transposition but should also guide the future fine-tuning of E. coli strains for gene cloning and protein expression, including phage display techniques, utilizing advanced tools for site-specific genome engineering.

Stem Cell Quiescence and Dormancy in Plant Meristems.

Plants exhibit opportunistic developmental patterns, alternating between growth and dormancy in response to external cues. Moreover, quiescence plays a critical role in proper plant growth and development, particularly within the root apical meristem (RAM) and the shoot apical meristem (SAM). In these meristematic tissues, cells with relatively slower mitotic activity are present in the quiescent center (QC) and the central zone (CZ), respectively. These centers form long-term reservoirs of stem cells maintaining the meristematic stem cell niche (SCN), and thus sustaining continuous plant development and adaptation to changing environments. This review explores the early observations, structural characteristics, functions, and gene regulatory networks of the RAM and SAM. It also highlights the intricate mechanism of dormancy within the SAM. The aim is to contribute to a holistic understanding of quiescence in plants, which is fundamental for the plant proper growth and environmental response.