Loss of fragile WWOX gene leads to senescence escape and genome instability.

Induction of DNA damage response (DDR) to ensure accurate duplication of genetic information is crucial for maintaining genome integrity during DNA replication. Cellular senescence is a DDR mechanism that prevents the proliferation of cells with damaged DNA to avoid mitotic anomalies and inheritance of the damage over cell generations. Human WWOX gene resides within a common fragile site FRA16D that is preferentially prone to form breaks on metaphase chromosome upon replication stress. We report here that primary Wwox knockout (Wwox-/-) mouse embryonic fibroblasts (MEFs) and WWOX-knockdown human dermal fibroblasts failed to undergo replication-induced cellular senescence after multiple passages in vitro. Strikingly, by greater than 20 passages, accelerated cell cycle progression and increased apoptosis occurred in these late-passage Wwox-/- MEFs. These cells exhibited γH2AX upregulation and microsatellite instability, indicating massive accumulation of nuclear DNA lesions. Ultraviolet radiation-induced premature senescence was also blocked by WWOX knockdown in human HEK293T cells. Mechanistically, overproduction of cytosolic reactive oxygen species caused p16Ink4a promoter hypermethylation, aberrant p53/p21Cip1/Waf1 signaling axis and accelerated p27Kip1 protein degradation, thereby leading to the failure of senescence induction in Wwox-deficient cells after serial passage in culture. We determined that significantly reduced protein stability or loss-of-function A135P/V213G mutations in the DNA-binding domain of p53 caused defective induction of p21Cip1/Waf1 in late-passage Wwox-/- MEFs. Treatment of N-acetyl-L-cysteine prevented downregulation of cyclin-dependent kinase inhibitors and induced senescence in Wwox-/- MEFs. Our findings support an important role for fragile WWOX gene in inducing cellular senescence for maintaining genome integrity during DDR through alleviating oxidative stress.

Genomic instabilities in hepatocellular carcinoma: biomarkers and application in immunotherapies.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers. For patients with advanced HCC, liver function decompensation often occurs, which leads to poor tolerance to chemotherapies and other aggressive treatments. Therefore, it remains critical to develop effective therapeutic strategies for HCC. Etiological factors for HCC are complex and multifaceted, including hepatitis virus infection, alcohol, drug abuse, chronic metabolic abnormalities, and others. Thus, HCC has been categorized as a "genomically unstable" cancer due to the typical manifestation of chromosome breakage and aneuploidy, and oxidative DNA damage. In recent years, immunotherapy has provided a new option for cancer treatments, and the degree of genomic instability positively correlates with immunotherapy efficacies. This article reviews the endogenous and exogenous causes that affect the genomic stability of liver cells; it also updates the current biomarkers and their detection methods for genomic instabilities and relevant applications in cancer immunotherapies. Including genomic instability biomarkers in consideration of cancer treatment options shall increase the patients' well-being.

Genome-wide meta-analysis identifies ancestry-specific loci for Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) is a devastating neurological disease with complex genetic etiology. Yet most known loci have only identified from the late-onset type AD in populations of European ancestry. METHODS: We performed a two-stage genome-wide association study (GWAS) of AD totaling 6878 Chinese and 63,926 European individuals. RESULTS: In addition to the apolipoprotein E (APOE) locus, our GWAS of two independent Chinese samples uncovered three novel AD susceptibility loci (KIAA2013, SLC52A3, and TCN2) and a novel ancestry-specific variant within EGFR (rs1815157). More replicated variants were observed in the Chinese (31%) than in the European samples (15%). In combining genome-wide associations and functional annotations, EGFR and TCN2 were prioritized as two of the most biologically significant genes. Phenome-wide Mendelian randomization suggests that high mean corpuscular hemoglobin concentration might protect against AD. DISCUSSION: The current study reveals novel AD susceptibility loci, emphasizes the importance of diverse populations in AD genetic research, and advances our understanding of disease etiology. HIGHLIGHTS: Loci KIAA2013, SLC52A3, and TCN2 were associated with Alzheimer's disease (AD) in Chinese populations. rs1815157 within the EGFR locus was associated with AD in Chinese populations. The genetic architecture of AD varied between Chinese and European populations. EGFR and TCN2 were prioritized as two of the most biologically significant genes. High mean corpuscular hemoglobin concentrations might have protective effects against AD.

Aerobic exercises regulate the epididymal anion homeostasis of high-fat diet-induced obese rats through TRPA1-mediated Cl- and HCO3- secretion†.

Aerobic exercises could improve the sperm motility of obese individuals. However, the underlying mechanism has not been fully elucidated, especially the possible involvement of the epididymis in which sperm acquire their fertilizing capacity. This study aims to investigate the benefit effect of aerobic exercises on the epididymal luminal milieu of obese rats. Sprague-Dawley male rats were fed on a normal or high-fat diet (HFD) for 10 weeks and then subjected to aerobic exercises for 12 weeks. We verified that TRPA1 was located in the epididymal epithelium. Notably, aerobic exercises reversed the downregulated TRPA1 in the epididymis of HFD-induced obese rats, thus improving sperm fertilizing capacity and Cl- concentration in epididymal milieu. Ussing chamber experiments showed that cinnamaldehyd (CIN), agonist of TRPA1, stimulated an increase of the short-circuit current (ISC) in rat cauda epididymal epithelium, which was subsequently abolished by removing the ambient Cl- and HCO3-. In vivo data revealed that aerobic exercises increased the CIN-stimulated Cl- secretion rate of epididymal epithelium in obese rats. Pharmacological experiments revealed that blocking cystic fibrosis transmembrane regulator (CFTR) and Ca2+-activated Cl- channel (CaCC) suppressed the CIN-stimulated anion secretion. Moreover, CIN application in rat cauda epididymal epithelial cells elevated intracellular Ca2+ level, and thus activate CACC. Interfering with the PGHS2-PGE2-EP2/EP4-cAMP pathway suppressed CFTR-mediated anion secretion. This study demonstrates that TRPA1 activation can stimulate anion secretion via CFTR and CaCC, which potentially forming an appropriate microenvironment essential for sperm maturation, and aerobic exercises can reverse the downregulation of TRPA1 in the epididymal epithelium of obese rats.

Dual role of sprouty2 as an inhibitor of RAS/ERK-driven proliferation and a promoter of cancer invasion in KRAS wild-type colorectal cancer.

Sprouty2 (SPRY2) is known to inhibit the RAS/MAPK/ERK pathway, and is a potential study target for cancer. The effect of SPRY2 in colorectal cancer (CRC) and whether it is influenced by KRAS mutation are not known. We manipulated SPRY2 gene expression and used an activating KRAS-mutant plasmid to determine its effect on CRC cell function in vitro and/or in vivo. We performed SPRY2 immunohistochemical staining in 143 CRC specimens and analyzed the staining results with various clinicopathological characteristics in relation to KRAS mutation status. SPRY2 knockdown in Caco-2 cells carrying the wild-type (WT) KRAS gene upregulated phosphorylated ERK (p-ERK) levels and increased cell proliferation in vitro, but inhibited cell invasion. However, SPRY2 knockdown in SW480 cells (activating KRAS mutant) or Caco-2 cells transfected with KRAS-mutant plasmid did not significantly alter p-ERK levels, cell proliferation, or invasion. The xenografts of SPRY2-knockdown Caco-2 cells were larger with less deep muscle invasion than those of control cells. The clinical cohort study revealed a positive association of SPRY2 protein expression with pT status, lymphovascular invasion, and perineural invasion in KRAS-WT CRCs. However, the associations were not observed in KRAS-mutant CRCs. Interestingly, high SPRY2 expression was related to shorter cancer-specific survival in both KRAS-WT and KRAS-mutant CRC patients. Our study demonstrated the dual role of SPRY2 as an inhibitor of RAS/ERK-driven proliferation and as a promoter of cancer invasion in KRAS-WT CRC. SPRY2 may promote the invasion and progression of KRAS-WT CRC, and might also enhance KRAS-mutant CRC progression through pathways other than invasion.

Dietary interventions for pediatric patients with functional abdominal pain disorders: a systematic review and network meta-analysis.

Dietary therapies are recommended for the treatment of pediatrics with functional abdominal pain disorders (FAPDs), but the comparative effectiveness among them is unclear. Therefore, the main aim of this systematic review and meta-analysis was to compare the effectiveness of differential dietary therapies in pediatrics with functional abdominal pain disorders. We searched PubMed, Embase, and the Cochrane Central Register of Controlled Trials databases from inception to February 28, 2023. Randomized clinical trials of dietary treatments for pediatric patients with functional abdominal pain disorders were included. The primary outcome was the improvement in abdominal pain. The secondary outcomes were changes in pain intensity and pain frequency. Thirty-one studies after screening 8695 retrieved articles were included, and 29 studies were available for network meta-analysis. Compared with placebo, fiber (RR, 4.86; 95%CI, 1.77 to 13.32; P-score = 0.84), synbiotics (RR, 3.92; 95%CI, 1.65 to 9.28; P-score = 0.75), and probiotics (RR, 2.18; 95%CI, 1.46 to 3.26; P-score = 0.46) had significantly larger effect on the improvement in abdominal pain, the three treatments had larger effect than placebo but statistically insignificant in difference in improving pain frequency and intensity. Similarly, there were no significant differences between the dietary treatments after indirect comparisons of the three outcomes.  Conclusion: Fiber supplements, synbiotics, and probiotics were efficacious in improving abdominal pain of FAPDs in children, suggested by very low or low evidence. The evidence of the efficacy of probiotics is more convincing than fiber and synbiotics when sample size and statistical power were considered. No difference in the efficacy of the three treatments. High-quality trials are needed to further investigate the efficacy of dietary interventions. What is Known: • Multiple dietary treatment options are available for functional abdominal pain disorders in the pediatric population, of which the most beneficial one is currently unknown. What is New: • This NMA found very low to low certainty of the evidence suggesting that fiber, synbiotics, and probiotics might be more efficacious in improving abdominal pain of FAPDs in children than the other dietary treatments. • There were no significant differences between active dietary treatments for changes in abdominal pain intensity.

CspZ FH-Binding Sites as Epitopes Promote Antibody-Mediated Lyme Borreliae Clearance.

Transmitted by ticks, the bacterium Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD), the most common vector-borne disease in the Northern hemisphere. No effective vaccines are currently available. B. burgdorferi sensu lato produces the CspZ protein that binds to the complement inhibitor, factor H (FH), promoting evasion of the host complement system. We previously showed that while vaccination with CspZ did not protect mice from B. burgdorferi infection, mice can be protected after immunization with CspZ-Y207A/Y211A (CspZ-YA), a CspZ mutant protein without FH-binding activity. To further study the mechanism of this protection, herein we evaluated both poly- and monoclonal antibodies recognizing CspZ FH-binding or non-FH-binding sites. We found that the anti-CspZ antibodies that recognize the FH-binding sites (i.e., block FH-binding activity) eliminate B. burgdorferi sensu lato in vitro more efficiently than those that bind to the non-FH-binding sites, and passive inoculation with anti-FH-binding site antibodies eradicated B. burgdorferi sensu lato in vivo. Antibodies against non-FH-binding sites did not have the same effect. These results emphasize the importance of CspZ FH-binding sites in triggering a protective antibody response against B. burgdorferi sensu lato in future LD vaccines.

Potential millennial-scale avian declines by humans in southern China.

Mounting observational records demonstrate human-caused faunal decline in recent decades, while accumulating archaeological evidence suggests an early biodiversity impact of human activities during the Holocene. A fundamental question arises concerning whether modern wildlife population declines began during early human disturbance. Here, we performed a population genomic analysis of six common forest birds in East Asia to address this question. For five of them, demographic history inference based on 25-33 genomes of each species revealed dramatic population declines by 4- to 48-fold over millennia (e.g. 2000-5000 thousand years ago). Nevertheless, summary statistics detected nonsignificant correlations between these population size trajectories and Holocene temperature variations, and ecological niche models explicitly predicted extensive range persistence during the Holocene, implying limited demographic consequence of Holocene climate change. Further analyses suggest high negative correlations between the reconstructed population declines and human disturbance intensities and indicate a potential driver of human activities. These findings provide a deep-time and large-scale insight into the recently recognized avifaunal decline and support an early origin hypothesis of human effects on biodiversity. Overall, our study sheds light on the current biodiversity crisis in the context of long-term human-environment interactions and offers a multi-evidential framework for quantitatively assessing the ecological consequences of human disturbance.

Yeast-expressed recombinant SARS-CoV-2 receptor binding domain RBD203-N1 as a COVID-19 protein vaccine candidate.

SARS-CoV-2 protein subunit vaccines are currently being evaluated by multiple manufacturers to address the global vaccine equity gap, and need for low-cost, easy to scale, safe, and effective COVID-19 vaccines. In this paper, we report on the generation of the receptor-binding domain RBD203-N1 yeast expression construct, which produces a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity was evaluated in mice. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized to be a homogeneous monomer that showed a well-defined secondary structure, was thermally stable, antigenic, and when adjuvanted on Alhydrogel in the presence of CpG it was immunogenic and induced high levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. The characteristics of the RBD203-N1 protein-based vaccine show that this candidate is another well suited RBD-based construct for technology transfer to manufacturing entities and feasibility of transition into the clinic to evaluate its immunogenicity and safety in humans.

Economic Analysis of Exclusionary EGFR Test Versus Up-Front NGS for Lung Adenocarcinoma in High EGFR Mutation Prevalence Areas.

BACKGROUND: This study sought to determine whether exclusionary EGFR mutation testing followed by next-generation sequencing (NGS) is a cost-efficient and timely strategy in areas with high prevalence rates of EGFR mutation. METHODS: We developed a decision tree model to compare exclusionary EGFR testing followed by NGS and up-front NGS. Patients entered the model upon diagnosis of metastatic lung adenocarcinoma. Gene alterations with FDA-approved targeted therapies included EGFR, ALK, ROS1, BRAF, RET, MET, NTRK, and KRAS. Model outcomes were testing-related costs; time-to-test results; monetary loss, taking both costs and time into consideration; and percentage of patients who could be treated by FDA-approved therapies. Stacked 1-way and 3-way sensitivity analyses were performed. RESULTS: Exclusionary EGFR testing incurred testing-related costs of US $1,387 per patient, a savings of US $1,091 compared with the costs of up-front NGS. The time-to-test results for exclusionary EGFR testing and up-front NGS were 13.0 and 13.6 days, respectively. Exclusionary EGFR testing resulted in a savings of US $1,116 in terms of net monetary loss, without a reduction of patients identified with FDA-approved therapies. The EGFR mutation rate and NGS cost had the greatest impact on minimizing monetary loss. Given that the tissue-based NGS turnaround time was shortened to 7 days, up-front NGS testing would become the best strategy if its price could be reduced to US $568 in Taiwan. CONCLUSIONS: In areas with high prevalence rates of EGFR mutation, exclusionary EGFR testing followed by NGS, rather than up-front NGS, is currently a cost-efficient strategy for metastatic lung adenocarcinoma.