Successive Generations Research Articles

Research of recombinant influenza A virus as a vector for Mycoplasma pneumoniae P1a and P30a.

Mycoplasma pneumoniae (MP) is a common respiratory pathogen affecting the longevity of the elderly and the health of children. However, the human vaccine against MP has not been successfully developed till now due to the poor immunogenicity and side effects of MP inactivated or attenuated vaccine. Therefore, it is necessary to develop a MP genetic engineering vaccine with influenza virus strain as vector. In this study, the major antigen genes P1a of MP adhesion factor P1(3862-4554 bases) and P30a of P30(49-822 bases) were inserted into the nonstructural protein (NS) gene of Influenza A virus strain A/Puerto Rio/8/34(H1N1), PR8 for short, to construct the recombinant vectors NS-P1a or NS-P30a. The recombinant pHW2000 plasmids containing NS-P1a or NS-P30a were cotransfected with the rest 7 fragments of PR8 into HEK293T cells. After inoculating chicken embryos, the recombinant influenza viruses rFLU-P1a and rFLU-P30a were rescued. RT-PCR and sequencing were used to identify the recombinant viruses. The hemagglutination titers of rFLU-P1a and rFLU-P30a were determined after five successive generations in chicken embryos so as to indicate the genetic stability of the recombinant viruses. The morphology of recombinant influenza viruses was observed under electron microscopy. P1a or P30a was designed to be inserted into the modified NS gene sequence separately and synthesized successfully. RT-PCR identification of the recombinant viruses rFLU-P1a and rFLU-P30a showed that P1a (693 bp), P30a (774 bp), NS-P1a (1992bp) and NS-P30a (2073 bp) bands were found, and the sequencing results were correct. After five successive generations, each virus generation has a certain hemagglutination titer (from 1:32 to 1:64), and the band of P1a or P30a can be seen in the corresponding positions. The virus particles under the electron microscope appeared as spheres or long strips connected by several particles, revealing a complete viral membrane structure composed of virus lipid bilayer, hemagglutinin, neuraminidase, and matrix proteins. The recombinant viruses rFLU-P1a and rFLU-P30a which carried the advantaged immune regions of the P1 and P30 genes in MP were successfully constructed and identified. And the genetic stability of rFLU-P1a or rFLU-P30a was relatively high. The typical and complete morphology of influenza virus was observed under the electron microscope. Our research provided a foundation for the further development of MP vaccines for human.

Tropical peat soil changes across successive oil palm generations in Sarawak, Malaysia

Oil palm is commonly replanted once reaching the end of its productive lifespan. This cyclical planting practice in oil palm plantations could have long-term implications for the humification and properties of tropical peat soil. This study aimed to investigate the changes observed across successive generations of oil palm plantations in Sarawak, Malaysia. Fourier Transform Infrared Spectroscopy (FTIR) was applied to examine the quality of the Soil Organic Matter (SOM), specifically the functional groups, humification index, Hydrophobicity Index (HI), and Degree of Degradation (DDI). Overall, the peat humification trend was in the order of 2nd Gen > Forest >1st Gen. The higher presence of recalcitrant compounds of lignin in the soil was attributed to the higher HI and lower DDI in the 2nd Gen. The relationship between the Pyrophosphate Solubility Index (PSI) and the humification index further revealed a significant increase in the relative abundance of humic substances with the maturity of degraded organic matter. These findings suggest a notable transition, implicating a shift towards a more stable form of SOM over the long-term utilization of tropical peatland for oil palm plantations. This is characterised by a significant increase in the relative abundance of aromatic, phenolic, and carboxylic functional groups. The study also highlights the need for further research on the linkage between these changes and greenhouse gas emissions to enhance our understanding of the long-term biogeochemical cycle of oil palm on tropical peatlands.

Number of Blastodisc Cells During the Laying Period for Two Successive Generations of Layer and Broiler Breeder.

In the poultry industry, genetic selection for growth performance is associated with poor reproductive efficiency and an increase in embryo mortality. The identification of new biomarkers is essential to improve these parameters. The blastodisc, composed of blastodermal cells, undergoes cellular events to achieve embryo development. Factors such as hen's age, temperature and time of egg storage could influence the number of blastodermal cells and impair embryo development. In this study, we investigated the variability of the number of viable cells of blastodisc (NVCB) that could be dependent on the stage of laying and on the breed and potentially associated with reproductive parameters. In experimental breeds, eggs were collected during the whole cycle of laying. Then, the protocol was repeated on industrial breeds (breeder hens) during five successive days at three stages of laying (before, after laying peak and at the end of laying period) for two generations (mothers and offsprings). For each egg, the blastodisc was dissected in order to count viable cells. For both experiments, the NVCB increased during the laying cycle. The NVCB was higher in broiler blastodisc compared to layer blastodisc for both generations. For layer breed, the NVCB were negatively correlated with laying rate for the first generation while positively associated for offsprings. However, the NVCB was positively correlated with laying rates in both generations for broiler hens and with fertility and hatchability rates. The NVCB from fresh oviposited fertilised eggs could be a potential tool in predicting on reproductive performances in poultry.

Effect of microplastics on the demography of Brachionus calyciflorus Pallas (Rotifera) over successive generations

Microplastics (MPs) are becoming increasingly common in freshwater ecosystems. Rotifers may involuntarily ingest MPs through non-selective filtration, thus causing non-real satiety, leading to adverse effects on their demography. Here, we evaluated the acute and chronic effects of MPs (30 µm) on two successive generations of Brachionus calyciflorus s.s. at 20 and 25 °C. Demographic variables and ingestion rates were compared in the absence and presence of microplastics (550 spheres mL-1). For the life table experiments, cohorts of twenty neonates were introduced into 20 mL medium with four replicates per treatment. Ingestion rates of the rotifer were tested at both test temperatures in the absence and presence of the microplastics in 20 mL containers with 1 ind. mL-1 per container. The rotifer population growth rates ranged from 0.55 to 1.05 d-1 depending on the treatment. MPs decreased the fecundity by 20–24 %. The average lifespan of rotifers exposed to MPs in the F0 generation was lower than that in the F1 generation at both temperatures, indicating that pre-exposure of mothers may offer some protection to the offspring. The population growth rate was higher at 25 °C. The ingestion rates were significantly lower in the presence of the microplastics. Our observations indicated that this strain of B. calyciflorus was better adapted to 20 °C; microplastics and higher test temperature resulted in a pronounced changes in the demographic variables of the rotifer.

Melatonin rescues pregnant female mice and their juvenile offspring from high fat diet-induced alzheimer disease neuropathy

High fat diet (HFD) is a prime factor, which contributes to the present epidemic of metabolic syndrome. Prolonged intake of HFD induces oxidative stress (OS) that in turn causes neuroinflammation, neurodegeneration, insulin resistance, amyloid burden, synaptic dysfunction and cognitive impairment hence leading to Alzheimer's disease neuropathy. Melatonin (secreted by the Pineal gland) has the potential to nullify the toxic effects of reactive oxygen species (ROS) and have been shown to ameliorate various complications induced by HFD in rodent models. This study aimed to assess the neurotherapeutic effects of melatonin on HFD-induced neuroinflammation and neurodegeneration mediated by OS in pregnant female mice and their offspring. Western blotting, immunohistochemistry and antioxidant enzyme assays were used for quantification of samples from the hippocampal region of the brain of pregnant albino mice and their offspring. Short- and long-term memory was assessed by Y-maze and Morris Water Maze tests. HFD significantly induced OS leading to AD like neuropathology in the pregnant mice and their offspring while melatonin administration simultaneously with the HFD significantly prevented this neuropathy. This study reports that melatonin exerts these effects through the stimulation of SIRT1/Nrf2/HO-1 pathway that in turn reduces the HFD-induced OS and its downstream signaling. In conclusion melatonin prevents HFD-induced multiple complications that ultimately leads to the memory dysfunction in pregnant female mice and their successive generation via activation of SIRT1/Nrf2 signaling pathway.

Electroporation Delivery of Cas9 sgRNA Ribonucleoprotein-Mediated Genome Editing in Sheep IVF Zygotes.

The utilization of electroporation for delivering CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.

Dissociation of Human and Mouse Tumor Tissue Samples for Single-cell RNA Sequencing.

Human tumor samples hold a plethora of information about their microenvironment and immune repertoire. Effective dissociation of human tissue samples into viable cell suspensions is a required input for the single-cell RNA sequencing (scRNAseq) pipeline. Unlike bulk RNA sequencing approaches, scRNAseq enables us to infer the transcriptional heterogeneity in tumor specimens at the single-cell level. Incorporating this approach in recent years has led to many discoveries, such as identifying immune and tumor cellular states and programs associated with clinical responses to immunotherapies and other types of treatments. Moreover, single-cell technologies applied to dissociated tissues can be used to identify accessible chromatin regions T and B cell receptor repertoire, and the expression of proteins, using DNA barcoded antibodies (CITEseq). The viability and quality of the dissociated sample are critical variables when using these technologies, as these can dramatically affect the cross-contamination of single cells with ambient RNA, the quality of the data, and interpretation. Moreover, long dissociation protocols can lead to the elimination of sensitive cell populations and the upregulation of a stress response gene signature. To overcome these limitations, we devised a rapid universal dissociation protocol, which has been validated on multiple types of human and murine tumors. The process begins with mechanical and enzymatic dissociation, followed by filtration, red blood lysis, and live dead enrichment, suitable for samples with a low input of cells (e.g., needle core biopsies). This protocol ensures a clean and viable single-cell suspension paramount to the successful generation of Gel Bead-In Emulsions (GEMs), barcoding, and sequencing.

Mesenchymal Vangl1 and Vangl2 facilitate airway elongation and widening independently of the planar cell polarity complex.

Adult mammalian lungs exhibit a fractal pattern, as each successive generation of airways is a fraction of the size of the parental branch. Achieving this structure likely requires precise control of airway length and diameter, as the embryonic airways initially lack the fractal scaling observed in the adult. In monolayers and tubes, directional growth can be regulated by the planar cell polarity (PCP) complex. Here, we characterized the roles of PCP complex components in airway initiation, elongation and widening during branching morphogenesis of the lung. Using tissue-specific knockout mice, we surprisingly found that branching morphogenesis proceeds independently of PCP complex function in the lung epithelium. Instead, we found a previously unreported Celsr1-independent role for the PCP complex components Vangl1 and Vangl2 in the pulmonary mesenchyme, where they are required for branch initiation, elongation and widening. Our data thus reveal an explicit function for Vangl1 and Vangl2 that is independent of the core PCP complex, suggesting a functional diversification of PCP complex components in vertebrate development. These data also reveal an essential role for the embryonic mesenchyme in generating the fractal structure of airways in the mature lung.

Biochemical and molecular characterization of pyrifluquinazon resistance in Bemisia tabaci Asia I

One of the most devastating polyphagous pests in the world, Bemisia tabaci (Gennadius), causes significant damage to a variety of crops and has demonstrated the ability to develop resistance to different classes of insecticides rapidly. A novel pyridine azomethine derivative pyrifluquinazon exhibits exceptional insecticidal toxicity against B. tabaci by interrupting the function of chordotonal receptor neurons. Formulating resistance management strategies is crucial to ensure the long-term use of this insecticide for whitefly control; however, the characteristics and possible mechanisms of pyrifluquinazon resistance in B. tabaci remain unclear. By employing pyrifluquinazon selection for 22 successive generations, the pyrifluquinazon-resistant strain (PQZ-R) was generated from a laboratory-susceptible population of B. tabaci Asia I (Lab-WB) and exhibited 39.65-fold resistance to pyrifluquinazon. When the realized heritability (h2) of B. tabaci to pyrifluquinazon was assumed to be the laboratory-estimated value (h2 = 0.181) and the mortality was 70–90%, only 12.3–22.6 generations were expected to be required to obtain a 10-fold increase in pyrifluquinazon resistance. While there was no significant cross-resistance to cyantraniliprole, dinotefuran, flonicamid, flupyradifurone, pymetrozine, sulfoxaflor, or thiamethoxam, the PQZ-R strain displayed slight cross-resistance to afidopyropen (3.14-fold). Synergism tests indicated that piperonyl butoxide (PBO) inhibits (4.36-fold) pyrifluquinazon resistance in the PQZ-R strain. This impact may be attributed to enhanced detoxification (elevated 3.91-fold) mediated by cytochrome P450 monooxygenase (P450). Compared to Lab-WB, the PQZ-R strain exhibited no significant overexpression of the 13 published detoxification-related P450 genes from CYP303, CYP4, and CYP6 families in B. tabaci. The combined knowledge gained from this study will enable further investigations into the function of qualitative and quantitative variations in P450-encoded genes, leading to innovative approaches for efficiently managing B. tabaci.

Emerging threat of marine microplastics: Cigarette butt contamination on Yellow Sea beaches and the potential toxicity risks to rotifer growth and reproduction

Cigarette butts have become one of the most common and persistent forms of debris in marine coastal areas, where they pose significant toxicity risks. This study investigated cigarette butt pollution along beaches of the Yellow Sea and used laboratory experiments to assess the toxicity of their leachate and fibers on the euryhaline rotifer Brachionus plicatilis. A pollution index confirmed pollution by this debris across all eight beaches surveyed, where the density of cigarette butts averaged 0.23 butts/m2. In controlled laboratory experiments, both the fibers and leachates from cigarette butts exhibited negative impacts on the development, reproduction, and population growth of rotifers. Unique abnormalities observed under different exposure treatments indicated toxicity specific to certain chemicals and particles. Continuous exposure to cigarette butts initially reduced rotifer fecundity, but this effect diminished over successive generations. However, the exposure induced transgenerational reproductive toxicity in the rotifers. Adaptive responses in rotifers after repeated exposure led to relative reduction in reproductive inhibition in the F3 and F4 generations. Furthermore, rotifers were capable of ingesting and accumulating cigarette butts, and maternal transfer emerged as an alternative pathway for uptake of this material in the offspring. These results increase our understanding of the ecological risks posed by cigarette butts in aquatic environments.

Deregulation of mitochondrial gene expression in cancer: mechanisms and therapeutic opportunities.

"Reprogramming of energy metabolism" was first considered an emerging hallmark of cancer in 2011 by Hanahan & Weinberg and is now considered a core hallmark of cancer. Mitochondria are the hubs of metabolism, crucial for energetic functions and cellular homeostasis. The mitochondrion's bacterial origin and preservation of their own genome, which encodes proteins and RNAs essential to their function, make them unique organelles. Successful generation of mitochondrial gene products requires coordinated functioning of the mitochondrial 'central dogma,' encompassing all steps necessary for mtDNA to yield mitochondrial proteins. Each of these processes has several levels of regulation, including mtDNA accessibility and protection through mtDNA packaging and epigenetic modifications, mtDNA copy number through mitochondrial replication, mitochondrial transcription through mitochondrial transcription factors, and mitochondrial translation through mitoribosome formation. Deregulation of these mitochondrial processes in the context of cancers has only recently been appreciated, with most studies being correlative in nature. Nonetheless, numerous significant associations of the mitochondrial central dogma with pro-tumor phenotypes have been documented. Several studies have even provided mechanistic insights and further demonstrated successful pharmacologic targeting strategies. Based on the emergent importance of mitochondria for cancer biology and therapeutics, it is becoming increasingly important that we gain an understanding of the underpinning mechanisms so they can be successfully therapeutically targeted. It is expected that this mechanistic understanding will result in mitochondria-targeting approaches that balance anticancer potency with normal cell toxicity. This review will focus on current evidence for the dysregulation of mitochondrial gene expression in cancers, as well as therapeutic opportunities on the horizon.

Performance of Artificial Diets for Zelus renardii (Hemiptera: Reduviidae) Rearing.

Mass production is a prerequisite for using natural enemies in integrated pest management and organic farming. Natural enemies in agroecosystems include predators that prey on insects, which they can subdue while maintaining adequate pest population densities. The Leafhopper Assassin Bug (LAB), Zelus renardii, can be a natural enemy in agroecosystems, selecting its prey for size and mobility. Some of LAB's prey include Philaenus spumarius (L.), Bactrocera oleae (Rossi), Drosophila suzukii (Matsumura), and Macrohomotoma gladiata Kuwayama, suggesting this reduviid for biocontrol agent in various contexts. We reared LABs for two subsequent broods offering living prey and artificial diets. Our data show that the rearing of Z. renardii is feasible with oligidic, meridic, and holidic artificial formulations. Four artificial diets allowed the complete post-embryonic development of LABs in captivity for two successive generations. The accumulated degree-days (ADDs) accurately predict the growth of LABs based on heat accumulation, estimating that up to three generations could grow per year in captivity at the experimented T°C.

Generation of induced pluripotent stem cells from the Asian bats

ABSTRACT Preservation of native Korean bats is crucial for maintaining ecological balance, as they play a vital role in insect control, pollination, and seed dispersal within their ecosystems. The present study details the establishment of bat induced pluripotent stem cells (BatiPSCs) from two Asian and Korean bats (Hypsugo alaschanicus and Pipistrellus abramus) using the Sendai Reprogramming Kit. Colonies of BatiPSCs, exhibiting distinctive features, were manually selected and expanded following successful transfection. Characterization of BatiPSCs revealed the expression of pluripotency markers, such as Octamer-binding transcription factor 4 (Oct4), SRY (sex-determining region Y)-box 2 and Nanog, with notably increased Oct4 levels and reduced Myc proto-oncogene expression compared with those noted in other induced pluripotent stem cell sources. BatiPSCs displayed positive staining for alkaline phosphatase and demonstrated the ability to form embryoid bodies, while also inducing teratomas in non-immune nude mice. Additionally, green fluorescent protein (GFP)-expressing BatiPSCs were generated and used for chimeric mouse production, with slight GFP signals detected in the neck region of the resulting mouse foetuses. These findings demonstrate the successful generation and characterization of BatiPSCs, emphasizing their potential applications in chimeric animal models, and the protection of endangered bat species.

Synthesis and characterization of hierarchically porous carbon anodes from furfural biorefinery residues for sustainable lithium-ion batteries

This study aims to valorize abundant lignocellulosic residues from furfural biorefineries into hierarchically porous carbon anodes for sustainable lithium-ion batteries (LIBs). Simultaneously, it addresses the growing demand for high-performance anode materials by leveraging renewable waste feedstocks. Sugarcane bagasse waste residues obtained after furfural extraction were used as the precursor. The furfural-derived bagasse residue (FDBR) underwent pyrolysis at 700 °C to produce a carbon-rich char, which was further activated using KOH and steam at 900 °C for 15–60 minutes to enhance porosity development. Comprehensive physicochemical characterizations, including proximate analysis, N2 adsorption-desorption, SEM-EDX, FTIR, and TGA/DTG, and Raman spectroscopy were performed to evaluate the properties of the synthesized activated carbons. The results demonstrated the successful generation of high surface area (405.8 m2/g) activated carbons with a hierarchical pore structure comprising micropores and mesopores. SEM and EDX analyses revealed a uniform, porous morphology enriched with 10.41 % at oxygen-containing functional groups. FTIR spectra confirmed the presence of hydroxyls, carbonyls, and carboxylic acid groups, beneficial for pseudocapacitive charge storage. The Raman analysis revealed a highly disordered and amorphous carbon structure with a significant concentration of defects, as evidenced by a high-intensity ratio (ID/IG ≈ 1.2–1.4) of the disordered carbon band to the graphitic band. The activated carbons exhibited a high initial discharge capacity of 1055 mAh/g in lithium half-cells, significantly exceeding the theoretical limit of commercial graphite anodes (372 mAh/g). This superior capacity was attributed to the high surface area, hierarchical porosity, and pseudocapacitive contributions from the oxygen functionalities. However, rapid capacity fading from 1055 to ∼250 mAh/g was observed over 100 cycles at a C/2 rate. The capacity retention stabilized at ∼88 % after 47 cycles, indicating irreversible capacity losses. These limitations were ascribed to the disordered amorphous structure, instability of the solid-electrolyte interphase, lithium trapping by oxygen groups, and structural changes during cycling. Optimizing activation conditions, incorporating conductive additives/coatings, exploring alternative binders, and surface functionalization are proposed to improve long-term cycling stability. Overall, this study demonstrates the feasibility of upcycling furfural biorefinery waste into sustainable LIB anodes while highlighting challenges and potential solutions for enhancing their electrochemical performance.

Adult Offspring of Deer Mouse Breeding Pairs Selected for Normal and Compulsive-Like Large Nesting Expression Invariably Show the Same Behavior Without Prior In-Breeding.

Obsessive-compulsive disorder is a neuropsychiatric condition with notable genetic involvement. Against this background, laboratory-housed deer mice of both sexes varyingly present with excessive and persistent large nesting behavior (LNB), which has been validated for its resemblance of clinical compulsivity. Although LNB differs from normal nesting behavior (NNB) on both a biological and cognitive level, it is unknown to what extent the expression of LNB and NNB is related to familial background. Here, we randomly selected 14 NNB- and 14 LNB-expressing mice (equally distributed between sexes) to constitute 7 breeding pairs of each phenotype. Pairs were allowed to breed two successive generations of offspring, which were raised until adulthood (12 weeks) and assessed for nesting expression. Remarkably, our findings show that offspring from LNB-expressing pairs build significantly larger nests compared to offspring from NNB-expressing pairs and the nesting expression of the offspring of each breeding pair, irrespective of parental phenotype or litter, is family specific. Collectively, the results of this investigation indicate that LNB can be explored for its potential to shed light on heritable neurocognitive mechanisms that may underlie the expression of specific persistent behavioral phenotypes.

A Review on Mastitis in Dairy Cows Research: Current Status and Future Perspectives

One of the most serious diseases affecting dairy cattle, causing significant losses both in breeding and economy, is mastitis, an inflammation of the mammary gland. Due to the economic importance of this issue, many research teams are striving to develop an easy-to-apply and, most importantly, effective method to prevent mastitis. The use of traditional methods for mastitis detecting and treating, as well as improvement in hygienic conditions, have not yielded the expected results in combating this disease combating. Currently, the main task is to find the tools that would allow for the rapid detection of mastitis and the improvement of udder health in cows while maintaining high milk production, which is essential for the profitability of dairy cattle farming. Accurate and rapid diagnostic tools, with the simultaneous capability of identifying pathogens, may help to reduce losses. Sufficient sensitivity and specificity for tests are required to minimize the number of false-positive and false-negative cases. Efforts are also being made to determine the optimal threshold value for detecting the disease at its earliest possible stage. The estimation of somatic cell count (SCC) as a phenotypic indicator of mastitis is widely used. A more precise parameter for accurately describing udder health is the differential somatic cell count (DSCC). The well-known California Mastitis Test (CMT) is an inexpensive, easy, and rapid method for mastitis detection useful on farms. The latest diagnostic methods for mastitis utilize tests based on the activity of N-acetyl-β-d-glucosaminidase (NAGase) or lactate dehydrogenase (LDH) as well as the determination of acute phase proteins (APPs) in blood serum and milk (such as haptoglobin, serum amyloid A, fibrinogen, and ceruloplasmin). Research also focuses on the genomic improvement of mastitis resistance in successive generations, and for this purpose, many quantitative trait loci (QTLs) and single nucleotide polymorphisms (SNPs) have been identified. In recent years, immunotherapy has become an increasingly common area of research, including vaccinations, T/B cell immunotherapy, RNA immunotherapy, epigenetic immunotherapy, stem cell therapy, and native secretory factors. An important aspect of the control of mastitis is the implementation of strategies that focus primarily on preventing the disease through appropriate breeding and farm management practices. In the forthcoming years, a significant challenge will be the development of universal diagnostic and therapeutic strategies that can be effectively implemented as alternatives to antibiotic therapy. Future research should prioritize the advancement of preventive and therapeutic techniques, such as immunotherapies, bacteriocins, herbal therapy, and nanoparticle technology.

Abstract We006: Metabolic Reprogramming to Increase Mitochodnrial Mass, Fusion and Energetics Represents an Important Rate Limiting Step in Direct Cardiac Reprogramming

Direct reprogramming of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) shows promise for treating myocardial infarction (MI). However, low conversion efficiency and relative immaturity of iCMs have so far precluded its clinical translation. Native CM maturation and cell cycle exit is associated with a switch from glycolytic metabolism to mitochondrial fatty acid oxidation (FAO), accompanied by a shift in the dynamic balance of mitochondrial fission (dividing) and fusion (joining) toward fusion. Because CFs lack the energy requirements of CMs, we hypothesized that transdifferentiation of CFs to iCMs requires a similar switch. Combining confocal microscopy with metabolic flux assays, we directly compared native CF and CM mitochondrial morphology and energetics. CMs showed significantly higher mitochondrial mass, fusion and energetics, all metrics relevant for increased reliance on mitochondrial FAO. Applying these techniques to study reprogramming, we found that at later reprogramming time points key metrics move significantly toward the phenotypes of native CMs. We then hypothesized that facilitating metabolic reprogramming by inhibiting fission and/or promoting fusion could improve iCM conversion. A loss of function screen identified certain fission genes, particularly the little studied mitochondrial fission regulator 1-like protein (Mtfr1l), as potent barriers to iCM conversion and maturation. In contrast, the core fusion genes were necessary for successful generation of iCMs, particularly the key fusion effector optical atrophy 1 (Opa1). Mtfr1l has been shown to promote cleavage of Opa1, and indeed concurrent knockdown of Opa1 ablated improvements to reprogramming efficiency. In conclusion, we showed both that iCMs are associated with increased mitochondrial mass, fusion and energetics approaching those of native CMs and that inducing CM-like mitochondria by knocking down Mtfr1l dramatically improved reprogramming, potentially through increased fusion activity of Opa1. By incorporating metabolic reprogramming into traditional direct cardiac reprogramming strategies, we may be able to move the field closer to clinical application to improve outcomes after MI.

Comprehensive Analysis of a Japanese Pedigree with Biallelic ACAGG Expansions in RFC1 Manifesting Motor Neuronopathy with Painful Muscle Cramps.

Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is an autosomal recessive multisystem neurologic disorder caused by biallelic intronic repeats in RFC1. Although the phenotype of CANVAS has been expanding via diagnostic case accumulation, there are scant pedigree analyses to reveal disease penetrance, intergenerational fluctuations in repeat length, or clinical phenomena (including heterozygous carriers). We identified biallelic RFC1 ACAGG expansions of 1000 ~ repeats in three affected siblings having sensorimotor neuronopathy with spinocerebellar atrophy initially presenting with painful muscle cramps and paroxysmal dry cough. They exhibit almost homogeneous clinical and histopathological features, indicating motor neuronopathy. Over 10years of follow-up, painful intractable muscle cramps ascended from legs to trunks and hands, followed by amyotrophy and subsequent leg pyramidal signs. The disease course combined with the electrophysical and imagery data suggest initial and prolonged hyperexcitability and the ensuing spinal motor neuron loss, which may progress from the lumbar to the rostral anterior horns and later expand to the corticospinal tract. Genetically, heterozygous ACAGG expansions of similar length were transmitted in unaffected family members of three successive generations, and some of them experienced muscle cramps. Leukocyte telomere length assays revealed comparatively shorter telomeres in affected individuals. This comprehensive pedigree analysis demonstrated a non-anticipating ACAGG transmission and high penetrance of manifestations with a biallelic state, especially motor neuronopathy in which muscle cramps serve as a prodromal and disease progress marker. CANVAS and RFC1 spectrum disorder should be considered when diagnosing lower dominant motor neuron disease, idiopathic muscle cramps, or neuromuscular hyperexcitability syndromes.

Rising incidence of obesity-related cancers among younger adults in China: A population-based analysis (2007-2021).

Developing countries face an "obesity epidemic," particularly affecting children and younger adults. While obesity is a known risk factor for 12 types of cancer, primarily affecting older populations, its impact on younger generations is understudied. This study analyzed data from a population-based cancer registry covering 14.14 million individuals in China (2007-2021). We compared the incidence of obesity- and non-obesity-related cancers and applied an age-period-cohort model to estimate their impacts. Among 651,342 cancer cases, 48.47% were obesity related. The age-standardized incidence rates (ASRs) of the 12 obesity-related cancers increased annually by 3.6% (p<0.001), while ASRs for non-obesity-related cancers remained stable. Obesity-related cancers surged among younger adults, with rates rising across successive generations. The annual percentage of change decreased with age, from 15.28% for ages 25-29 years to 1.55% for ages 60-64 years. The incidence rate ratio for obesity-related cancer was higher in younger generations compared to those born in 1962-1966. We predict that the ASR for obesity-related cancers will nearly double in the next decade. The rising incidence of obesity-related cancers among young adults poses a significant public health concern. The increasing cancer burden underscores the need for targeted interventions to address the obesity epidemic. This work was supported by the National Natural Science Foundation of China (81930019, 82341076) to J.-K.Y.

Revisiting the Past: Historical Status of Breeding Ring-billed and California Gulls in the Klamath Basin, Oregon

Three summaries of the historical and recent status of breeding Ring-billed (Larus delawarensis) and California (L. californicus) gulls in Oregon mention just two colony sites in the Klamath Basin—Upper Klamath Lake and Spring Lake—and lack numerical data for individual colonies. Nevertheless, nesting of gulls at Lower Klamath Lake before its water source was cut off in 1917 is well documented. Egg-set records, published literature, and archived field notes attest to nesting of both species before 1954 at two additional sites in the Klamath Basin, the Link and Klamath rivers. The largest known colonies up to that time were at Lower Klamath Lake (~2000 pairs, perhaps many more) and along the Klamath River below the town of Klamath Falls (~2000 pairs). Gulls no longer nest at these five sites, but, since at least 2000, they have nested intermittently at Swan Lake and Gerber Reservoir. Though Gabrielson and Jewett (1940) considered the California Gull more abundant in southeastern Oregon than the Ring-billed, egg-collectors’ data from 1926 to 1953 suggest the opposite, as in northeastern California. The California Gull may have been the more numerous of the two in the Klamath Basin when nesting and foraging habitat was dominated by wetlands, but the drying of Lower Klamath Lake and the great expansion of irrigated agriculture starting in the 1920s may have favored the Ring-billed. Trends in the Klamath Basin’s gull populations are unclear, but much nesting habitat has been lost. Documentation of the historical record of nesting gulls and other waterbirds is critical, as conservation efforts typically aim at restoring populations to some historical baseline, which can gradually shift lower with further declines and successive generations accepting a new status quo.