Cytoplasmic Granules Research Articles

Intermolecular energy migration via homoFRET captures the modulation in the material property of phase-separated biomolecular condensates

Physical properties of biomolecular condensates formed via phase separation of proteins and nucleic acids are associated with cell physiology and disease. Condensate properties can be regulated by several cellular factors including post-translational modifications. Here, we introduce an application of intermolecular energy migration via homo-FRET (Förster resonance energy transfer), a nanometric proximity ruler, to study the modulation in short- and long-range protein-protein interactions leading to the changes in the physical properties of condensates of fluorescently-tagged FUS (Fused in Sarcoma) that is associated with the formation of cytoplasmic and nuclear membraneless organelles. We show that homoFRET captures modulations in condensate properties of FUS by RNA, ATP, and post-translational arginine methylation. We also extend the homoFRET methodology to study the in-situ formation of cytoplasmic stress granules in mammalian cells. Our studies highlight the broad applicability of homoFRET as a potent generic tool for studying intracellular phase transitions involved in function and disease.

Malakoplakia, a rare inflammatory condition presenting as a colon polyp

Abstract Introduction/Objective Malakoplakia is a rare histiocytic disease caused by defects in phagocytic or degradative functions of histiocytes in response to gram negative coliforms (E. coli or Proteus) that results in chronic inflammatory state, followed by intracellular deposition of iron and calcium (known as Michaelis-Gutmann bodies). It’s more common in immunocompromised patients and the mean age at diagnosis is fifth decade. It occurs in all organs, common in GU tract, particularly bladder, but also seen in gastrointestinal tract (most commonly colon, followed by stomach and duodenum), central nervous system and female genital tract. In gastrointestinal tract, it can present as a polyp with diffuse mucosal involvement, submucosal thickening, and even may cause mass. Methods/Case Report A 60 -year-old male smoker presented to our hospital with history of colon polyps; colonoscopy revealed a 6 mm sessile polyp at the hepatic flexure. A biopsy was performed which showed numerous epithelioid histiocytes with granular eosinophilic cytoplasm, abundant Michaelis-Gutmann bodies and increased lymphocytes. Immunohistochemical stains showed diffuse positivity for CD68; and PAS stain was positive for histiocytic cytoplasmic granules. Based on this data, the diagnosis of Malakoplakia was confirmed. Malakoplakia is treated with antibiotics that concentrate in macrophages (quinolones or trimethoprim-sulfamethoxazole), antibiotics directed against E. coli, surgery and possibly discontinuation of immunosuppressive drug therapy. Results (if a Case Study enter NA) Malakoplakia, in this case, was presented clinically as a sessile colon polyp and histologically showed numerous epithelioid histiocytes with granular eosinophilic cytoplasm, with Immunohistochemical stains (CD68; and PAS stain) the correct diagnosis was made. Other presentations of Malakoplakia include diffuse mucosal involvement, submucosal thickening, or mass. Histologically, it may rarely be mistaken as signet ring carcinoma. These variable presentations in GU tract or colon can be interpreted as malignancy with consequent unnecessary interventions. Hence, awareness of Malakoplakia with careful histopathologic examination and the use of immunohistochemical studies are advisable in that cases. Conclusion We would like to emphasize that Malakoplakia can be clinically and histologically misdiagnosed as a malignant condition; therefore histopathologic and immunohistochemical studies are needed to correctly diagnose this entity.

A role for BYN-1/bystin in cellular uptake and clearance of residual bodies in the Caenorhabditis elegans germline.

GLH/Vasa/DDX4 helicases are core germ-granule proteins that promote germline development and fertility. A yeast-two-hybrid screen using Caenorhabditis elegans GLH-1 as bait identified BYN-1, the homolog of human bystin/BYSL. In humans, bystin promotes cell adhesion and invasion in gliomas, and, with its binding partner trophinin, triggers embryonic implantation into the uterine wall. C. elegans embryos do not implant and lack a homolog of trophinin, but both trophinin and GLH-1 contain unique decapeptide phenylalanine-glycine (FG)-repeat domains. In germ cells, we find endogenous BYN-1 in the nucleolus, partitioned away from cytoplasmic germ granules. However, BYN-1 enters the cytoplasm during spermatogenesis to colocalize with GLH-1. Both proteins become deposited in residual bodies (RBs), which are then engulfed and cleared by the somatic gonad. We show that BYN-1 acts upstream of CED-1 to drive RB engulfment, and that removal of the FG-repeat domains from GLH-1 and GLH-2 can partially phenocopy byn-1 defects in RB clearance. These results point to an evolutionarily conserved pathway whereby cellular uptake is triggered by the cytoplasmic mobilization of bystin/BYN-1 to interact with proteins harboring FG-repeats.

Pericentriolar material 1 promotes intestinal inflammation in ulcerative colitis by activating NLRP3/gasdermin D‐mediated macrophage pyroptosis

AbstractExcessive proinflammatory cytokine release induced by pyroptosis plays a vital role in intestinal mucosal inflammation in ulcerative colitis (UC). Several pyroptosis‐related factors are regulated by the centrosome. Pericentriolar material 1 (PCM1) is a primary component of centriolar satellites that is present as cytoplasmic granules around the centrosome. Our previous study revealed that PCM1 was highly expressed in UC patients, but the role of PCM1 in UC remains unknown. This study aimed to elucidate the role of PCM1 in the development of UC, especially the mechanism in pyroptosis process of UC. Clinical mucosal sample and dextran sulfate sodium (DSS)‐induced colitis mouse were used to reveal the association between PCM1 and intestinal inflammation. Intestinal epithelial cell‐specific PCM1‐knockout mice were constructed to determine the role of PCM1 in colitis. Finally, PCM1 RNA interference and overexpression assays in THP1 cells were employed to study the molecular mechanisms of PCM1 in inflammatory responses and pyroptosis. We found that PCM1 expression was upregulated in the colonic mucosa of UC patients and positively correlated with inflammatory indicators. PCM1 expression was elevated in DSS‐induced colitis mice and was reduced after methylprednisolone treatment. In the DSS colitis model, intestinal‐specific PCM1‐knockout mice exhibited milder intestinal inflammation and lower pyroptosis levels than wild‐type mice. In cell level, PCM1 exerted a proinflammatory effect by activating the NLRP3 inflammasome and triggering subsequent gasdermin D‐mediated pyroptosis to release IL‐1β and IL‐18. In conclusion, PCM1 mediates activation of the NLRP3 inflammasome and gasdermin D‐dependent pyroptosis, ultimately accelerating intestinal inflammation in UC. These findings revealed a previously unknown role of PCM1 in initiating intestinal mucosal inflammation and pyroptosis in UC, and this factor is expected to be a regulator in the complex inflammatory network of UC.

Modeling aging and retinal degeneration with mitochondrial DNA mutation burden.

Somatic mitochondrial DNA (mtDNA) mutation accumulation has been observed in individuals with retinal degenerative disorders. To study the effects of aging and mtDNA mutation accumulation in the retina, a polymerase gamma (POLG) exonuclease-deficient model, the PolgD257A mutator mice (D257A), was used. POLG is an enzyme responsible for regulating mtDNA replication and repair. Retinas of young and older mice with this mutation were analyzed invivo and exvivo to provide new insights into the contribution of age-related mitochondrial (mt) dysfunction due to mtDNA damage. Optical coherence tomography (OCT) image analysis revealed a decrease in retinal and photoreceptor thickness starting at 6 months of age in mice with the D257A mutation compared to wild-type (WT) mice. Electroretinography (ERG) testing showed a significant decrease in all recorded responses at 6 months of age. Sections labeled with markers of different types of retinal cells, including cones, rods, and bipolar cells, exhibited decreased labeling starting at 6 months. However, electron microscopy analysis revealed differences in retinal pigment epithelium (RPE) mt morphology beginning at 3 months. Interestingly, there was no increase in oxidative stress and parkin-mediated mitophagy in the ages analyzed in the retina or RPE of D257A mice. Additionally, D257A RPE exhibited an accelerated rate of autofluorescence cytoplasmic granule formation and accumulation. Mt markers displayed different abundance in protein lysates obtained from retina and RPE samples. These findings suggest that the accumulation of mtDNA mutations leads to impaired mt function and accelerated aging, resulting in retinal degeneration.

Redox-dependent condensation and cytoplasmic granulation by human ssDNA-binding protein-1 delineate roles in oxidative stress response

Human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) plays central roles in DNA repair. Here, we show that purified hSSB1 undergoes redox-dependent liquid-liquid phase separation (LLPS) in the presence of single-stranded DNA or RNA, features that are distinct from those of LLPS by bacterial SSB. hSSB1 nucleoprotein droplets form under physiological ionic conditions in response to treatment modeling cellular oxidative stress. hSSB1's intrinsically disordered region is indispensable for LLPS, whereas all three cysteine residues of the oligonucleotide/oligosaccharide-binding fold are necessary to maintain redox-sensitive droplet formation. Proteins interacting with hSSB1 show selective enrichment inside hSSB1 droplets, suggesting tight content control and recruitment functions for the condensates. While these features appear instrumental for genome repair, we detected cytoplasmic hSSB1 condensates in various cell lines colocalizing with stress granules upon oxidative stress, implying extranuclear function in cellular stress response. Our results suggest condensation-linked roles for hSSB1, linking genome repair and cytoplasmic defense.

A transcription factor ATF3 involves in the phagocytosis of granulocytes in oyster Crassostrea gigas

Phagocytosis is a major cellular mechanism for mollusk granulocytes to eliminate nonself substances and dead cells, and thus to preserve the immune homeostasis. The knowledge of the regulatory mechanisms controlling phagocytic capacity is vital to understanding the immune system. In the present study, an ATF3 homolog (CgATF3) with a typical bZIP domain was identified in the Pacific oyster Crassostrea gigas. Its highly conserved bZIP domain consisted of two structural features, a basic region for DNA binding and a leucine zipper region for dimerization. Its transcript was found to be abundantly expressed in haemocytes, which was induced by Vibrio splendidus stimulation and recombinant CgTNF-2 treatment, along with an increase of its protein content in the nucleus. Moreover, CgATF3 showed a consistent and specific high expression in granulocytes, and CgATF3+ granulocytes were characterized morphologically by the largest diameter, smaller nucleus to cytoplasmic ratio, and abundant cytoplasmic granules, and functionally by a higher capacity for phagocytosis. When CgATF3 expression was inhibited by RNAi, the expression levels of CgRab1, CgRab33 and CgCathepsin L1, as well as the phagocytic rate and index of granulocytes all decreased after V. splendidus stimulation. These results together demonstrated the involvement of CgATF3 in regulating the expressions of Rabs and Cathepsin L1, as well as the phagocytosis of granulocytes in oyster C. gigas.

SARS-CoV-2 nucleocapsid protein promotes self-deacetylation by inducing HDAC6 to facilitate viral replication

BackgroundThe global outbreak of COVID-19 caused by the SARS-CoV-2 has led to millions of deaths. This unanticipated emergency has prompted virologists across the globe to delve deeper into the intricate dynamicity of the host-virus interface with an aim to identify antiviral targets and elucidate host and viral determinants of severe disease.AimThe present study was undertaken to analyse the role of histone deacetylase 6 (HDAC6) in regulating SARS-CoV-2 infection.ResultsGradual increase in HDAC6 expression was observed in different SARS-CoV-2-permissive cell lines following SARS-CoV-2 infection. The SARS-CoV-2 nucleocapsid protein (N protein) was identified as the primary viral factor responsible for upregulating HDAC6 expression. Downregulation of HDAC6 using shRNA or a specific inhibitor tubacin resulted in reduced viral replication suggesting proviral role of its deacetylase activity. Further investigations uncovered the interaction of HDAC6 with stress granule protein G3BP1 and N protein during infection. HDAC6-mediated deacetylation of SARS-CoV-2 N protein was found to be crucial for its association with G3BP1.ConclusionThis study provides valuable insights into the molecular mechanisms underlying the disruption of cytoplasmic stress granules during SARS-CoV-2 infection and highlights the significance of HDAC6 in the process.

Nanoscale dynamics and localization of single endogenous mRNAs in stress granules.

Stress granules (SGs) are cytoplasmic messenger ribonucleoprotein granules transiently formed in stressed mammalian cells. Although SG components have been well characterized, detailed insights into the molecular behavior inside SGs remain unresolved. We investigated nanoscale dynamics and localization of endogenous mRNAs in SGs combining single mRNA tracking and super-resolution localization microscopy. First, we developed a methodology for tracking single mRNAs within SGs, revealing that although mRNAs in SGs are mainly stationary (∼40%), they also move in a confined (∼25%) or freely diffusing (∼35%) manner. Second, the super-resolution localization microscopy showed that the mRNAs in SGs are heterogeneously distributed and partially form high-density clusters. Third, we simultaneously performed single mRNA tracking and super-resolution microscopy in SGs, demonstrating that single mRNA trajectories are mainly found around high-density clusters. Finally, a quantitative analysis of mRNA localization and dynamics during stress removal was conducted using live super-resolution imaging and single-molecule tracking. These results suggest that SGs have a highly organized structure that enables dynamic regulation of the mRNAs at the nanoscale, which is responsible for the ordered formation and the wide variety of functions of SGs.

Human Neutrophil Elastase: Characterization of Intra- vs. Extracellular Inhibition.

Neutrophil elastase (HNE), like other members of the so-called GASPIDs (Granule-Associated Serine Peptidases of Immune Defense), is activated during protein biosynthesis in myeloid precursors and stored enzymatically active in cytoplasmic granules of resting neutrophils until secreted at sites of host defense and inflammation. Inhibitors thus could bind to the fully formed active site of the protease intracellularly in immature progenitors, in circulating neutrophils, or to HNE secreted into the extracellular space. Here, we have compared the ability of a panel of diverse inhibitors to inhibit HNE in the U937 progenitor cell line, in human blood-derived neutrophils, and in solution. Most synthetic inhibitors and, surprisingly, even a small naturally occurring proteinaceous inhibitor inhibit HNE intracellularly, but the extent and dynamics differ markedly from classical enzyme kinetics describing extracellular inhibition. Intracellular inhibition of HNE potentially affects neutrophil functions and has side effects, but it avoids competition of inhibitors with extracellular substrates that limit its efficacy. As both intra- and extracellular inhibition have advantages and disadvantages, the quantification of intracellular inhibition, in addition to classical enzyme kinetics, will aid the design of novel, clinically applicable HNE inhibitors with targeted sites of action.

Replication properties of a contemporary Zika virus from West Africa.

Zika virus (ZIKV) has become a global health problem over the past decade due to the extension of the geographic distribution of the Asian/American genotype. Recent epidemics of Asian/American ZIKV have been associated with developmental disorders in humans. There is mounting evidence that African ZIKV may be associated with increased fetal pathogenicity necessitating to pay a greater attention towards currently circulating viral strains in sub-Saharan Africa. Here, we generated an infectious molecular clone GUINEA-18 of a recently transmitted human ZIKV isolate from West Africa, ZIKV-15555. The available infectious molecular clone MR766MC of historical African ZIKV strain MR766-NIID was used for a molecular clone-based comparative study. Viral clones GUINEA-18 and MR766MC were compared for their ability to replicate in VeroE6, A549 and HCM3 cell lines. There was a lower replication rate for GUINEA-18 associated with weaker cytotoxicity and reduced innate immune system activation compared with MR766MC. Analysis of chimeric viruses between viral clones stressed the importance of NS1 to NS4B proteins, with a particular focus of NS4B on GUINEA-18 replicative properties. ZIKV has developed strategies to prevent cytoplasmic stress granule formation which occurs in response to virus infection. GUINEA-18 was greatly efficient in inhibiting stress granule assembly in A549 cells subjected to a physiological stressor, with NS1 to NS4B proteins also being critical in this process. The impact of these GUINEA-18 proteins on viral replicative abilities and host-cell responses to viral infection raises the question of the role of nonstructural proteins in the pathogenicity of currently circulating ZIKV in sub-Saharan Africa.

P-164 The impact of oocyte morphological abnormalities on fertilization rate and blastocyst quality

Abstract Study question Do distinct morphological abnormalities in oocytes have an adverse effect on both fertilization rates and the quality of resulting blastocyst? Summary answer While most oocyte morphological abnormalities did not significantly affect outcomes, dark cytoplasmic color correlated with lower fertilization rates and the absence of good quality blastocysts What is known already Variability exists in the fertilization and developmental potential of mature (metaphase II - MII) human oocytes. A high-quality MII oocyte is characterized by a spherical shape, homogeneous cytoplasm, smooth appearance, a uniform zona pellucida (ZP), a normal-sized perivitelline space (PVS), and a single unfragmented polar body (PB). Dysmorphisms, such as variations in cytoplasmic features and anomalies in PVS size and PB regularity may occur. There are conflicting reports on the relationship between oocyte morphology and IVF outcomes. A recent meta-analysis highlighted associations between poor fertilization potential and specific dysmorphisms, including large/irregular PB, a large PVS, refractile bodies, and vacuoles. Study design, size, duration This study analyzed 367 MII oocytes obtained from 40 young patients. The fertilization rate (FR) and blastocyst quality (BQ) were compared among 132 dysmorphic and 235 normal MII oocytes (from October 2023 to January 2024). Dysmorphic oocytes were categorized as having a non-spherical shape, large PVS, PVS debris, abnormal ZP, fragmented PB, excess cytoplasmic granules, vacuoles, and dark cytoplasmic color. Participants/materials, setting, methods All MII oocyte denuded were examined under the microscope at x-400 magnification. Dysmorphic and normal oocytes were separated, injected, and cultured individually until day 5 under conditions of 37oC and 6% CO2. Fertilization was assessed 14 to 18 hours post-injection, and subsequent embryonic development was evaluated on day 2 and day 5. Blastocysts graded 4AA to 4BC are considered good quality, while those graded 4CC to 3CC are classified as fair quality. Main results and the role of chance The mean age of patients was 24.14 ± 3.77. In total, 36% of all oocytes exhibit at least one dysmorphism (n = 132). From the overall dataset analyzed, there was no significant difference (p > 0.9999) in FR between the normal 77.87% (183/235) and dysmorphic group of oocytes 78.03 (103/132). The odd ratio and 95% confidence interval are 0.9909 and 0.5957 to 1.633 respectively. However, this was not the same for oocytes with dark color cytoplasm. The presence of this dysmorphism significantly lowered FR (p = 0.0176) with an odd ratio of 5.544 (95% CI: 1.419 to 21.67). Similarly, the result showed no statistically significant difference (p = 0.1073) in the likelihood of obtaining a fair quality blastocyst between the normal oocytes 23.40% (55/235) and dysmorphic group of oocytes 15.79 (21/133). This was also the same for the likelihood of obtaining good quality blastocyst. There was no statistically significant difference (p = 0.1536) between the normal group 20.00% (47/235) and the dysmorphic group 26.52 (35/132). Oocyte with dark color cytoplasm gave no good quality blastocyst. Limitations, reasons for caution The exclusive use of oocytes from young patients may limit the generalizability of findings to a broader population of IVF patients. Also, the sample size may limit the study’s ability to detect subtle associations. A reason for caution when interpreting this study is its focus on morphological assessments alone. Wider implications of the findings The knowledge from our findings may contribute to optimizing oocyte selection strategies for intracytoplasmic sperm injection. Also, the knowledge could potentially be integrated into AI modules for oocyte morphological assessment. However, more studies would be required to develop a robust and scalable system Trial registration number Not applicable

Biomarkers of Cellular Senescence and Aging: Current State-of-the-Art, Challenges and Future Perspectives.

Population aging has increased the global prevalence of aging-related diseases, including cancer, sarcopenia, neurological disease, arthritis, and heart disease. Understanding aging, a fundamental biological process, has led to breakthroughs in several fields. Cellular senescence, evinced by flattened cell bodies, vacuole formation, and cytoplasmic granules, ubiquitously plays crucial roles in tissue remodeling, embryogenesis, and wound repair as well as in cancer therapy and aging. The lack of universal biomarkers for detecting and quantifying senescent cells, in vitro and in vivo, constitutes a major limitation. The applications and limitations of major senescence biomarkers, including senescence-associated β-galactosidase staining, telomere shortening, cell-cycle arrest, DNA methylation, and senescence-associated secreted phenotypes are discussed. Furthermore, explore senotherapeutic approaches for aging-associated diseases and cancer. In addition to the conventional biomarkers, this review highlighted the in vitro, in vivo, and disease models used for aging studies. Further, technologies from the current decade including multi-omics and computational methods used in the fields of senescence and aging are also discussed in this review. Understanding aging-associated biological processes by using cellular senescence biomarkers can enable therapeutic innovation and interventions to improve the quality of life of older adults.

FUS Selectively Facilitates circRNAs Packing into Small Extracellular Vesicles within Hypoxia Neuron.

Small extracellular vesicles (sEVs) contain abundant circular RNAs (circRNAs) and are involved in cellular processes, particularly hypoxia. However, the process that packaging of circRNAs into neuronal sEVs under hypoxia is unclear. This study revealed the spatial mechanism of the Fused in Sarcoma protein (FUS) that facilitates the loading of functional circRNAs into sEVs in hypoxia neurons. It is found that FUS translocated from the nucleus to the cytoplasm and is more enriched in hypoxic neuronal sEVs than in normal sEVs. Cytoplasmic FUS formed aggregates with the sEVs marker protein CD63 in cytoplasmic stress granules (SGs) under hypoxic stress. Meanwhile, cytoplasmic FUS recruited of functional cytoplasmic circRNAs to SGs. Upon relief of hypoxic stress and degradation of SGs, cytoplasmic FUS is transported with those circRNAs from SGs to sEVs. Validation of FUS knockout dramatically reduced the recruitment of circRNAs from SGs and led to low circRNA loading in sEVs, which is also confirmed by the accumulation of circRNAs in the cytoplasm. Furthermore, it is showed that the FUS Zf_RanBP domain regulates the transport of circRNAs to sEVs by interacting with hypoxic circRNAs in SGs. Overall, these findings have revealed a FUS-mediated transport mechanism of hypoxia-related cytoplasmic circRNAs loaded into sEVs under hypoxic conditions.

Structural characteristics and regenerative potential: Insights from the molly fish spinal cord.

Unlike mammals, species such as fish and amphibians can regenerate damaged spinal cords, offering insights into potential therapeutic targets. This study investigates the structural features of the molly fish spinal cord through light and electron microscopy. The most notable characteristic was the presence of Mauthner cells (M-cells), which exhibited large cell bodies and processes, as well as synaptic connections with astrocytes. These astrocytic connections contained synaptic vesicles, suggesting electrical transmission at the M-cell endings. Astrocytes, which were labeled with glial fibrillary acidic protein (GFAP), contained cytoplasmic glycogen granules, potentially serving as an emergency fuel source. Two types of oligodendrocytes were identified: a small, dark cell and a larger, lighter cell, both of which reacted strongly with oligodendrocyte transcription factor 2 (Olig2). The dark oligodendrocyte resembled human oligodendrocyte precursors, while the light oligodendrocyte was similar to mature human oligodendrocytes. Additionally, proliferative neurons in the substantia grisea centralis expressed myostatin, Nrf2, and Sox9. Collectively, these findings suggest that the molly fish spinal cord has advanced structural features conducive to spinal cord regeneration and could serve as an excellent model for studying central nervous system regeneration. Further studies on the functional aspects of the molly fish spinal cord are recommended. RESEARCH HIGHLIGHTS: Mauthner cells (M-cell), with their typical large cell body and processes, were the most characteristic feature in Molly fish spinal cord, where it presented synaptic connections with astrocytes and their ends contained synaptic vesicles indicating an electrical transmission in the M-cells endings. Two types of oligodendrocytes could be recognized; both reacted intensely with Oligodendrocyte transcription factor 2 (Olig2). The proliferative neurons of the substantia grisea centralis expressed myostatin, Nrf2, and Sox9. The findings of this study suggest that molly fish possess highly developed structural features conducive to spinal cord regeneration. Consequently, they could be deemed an exemplary model for investigating central nervous system regeneration.

Snapper-Schneid bodies (coarse cytoplasmic granules) in a patient with multiply relapsed multiple myeloma

Snapper-Schneid bodies (coarse cytoplasmic granules) in a patient with multiply relapsed multiple myeloma

Network analysis combined with experimental assessment to explore the therapeutic mechanisms of New Shenqi Pills formula targeting mitochondria on senile diabetes mellitus.

The escalation of global population aging has accentuated the prominence of senile diabetes mellitus (SDM) as a consequential public health concern. Oxidative stress and chronic inflammatory cascades prevalent in individuals with senile diabetes significantly amplify disease progression and complication rates. Traditional Chinese Medicine (TCM) emerges as a pivotal player in enhancing blood sugar homeostasis and retarding complication onset in the clinical management of senile diabetes. Nonetheless, an evident research gap persists regarding the integration of TCM's renal tonification pharmacological mechanisms with experimental validation within the realm of senile diabetes therapeutics. The objective of this study was to investigate the mechanisms of action of New Shenqi Pills (SQP) in the treatment of SDM and make an experimental assessment. Network analysis is used to evaluate target pathways related to SQP and SDM. Mitochondrial-related genes were obtained from the MitoCarta3.0 database and intersected with the common target genes of the disease and drugs, then constructing a protein-protein interaction (PPI) network making use of the GeneMANIA database. Representative compounds in the SQP were quantitatively measured using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to ensure quality control and quantitative analysis of the compounds. A type 2 diabetes mice (C57BL/6) model was used to investigate the pharmacodynamics of SQP. The glucose lowering efficacy of SQP was assessed through various metrics including body weight and fasting blood glucose (FBG). To elucidate the modulatory effects of SQP on pancreatic beta cell function, we measured oral glucose tolerance test (OGTT), insulin histochemical staining and tunel apoptosis detection, then assessed the insulin-mediated phosphoinositide 3-kinase (PI3K)/protein kinase A (Akt)/glycogen synthase kinase-3β (GSK-3β) pathway in diabetic mice via Western blotting. Additionally, we observe the structural changes of the nucleus, cytoplasmic granules and mitochondria of pancreatic islet β cells. In this investigation, we identified a total of 1876 genes associated with senile diabetes, 278 targets of SQP, and 166 overlapping target genes, primarily enriched in pathways pertinent to oxidative stress response, peptide response, and oxygen level modulation. Moreover, an intersection analysis involving 1,136 human mitochondrial genes and comorbidity targets yielded 15 mitochondria-related therapeutic targets. Quality control assessments and quantitative analyses of SQP revealed the predominant presence of five compounds with elevated concentrations: Catalpol, Cinnamon Aldehyde, Rehmanthin D, Trigonelline, and Paeonol Phenol. Vivo experiments demonstrated notable findings. Relative to the control group, mice in the model group exhibited significant increases in body weight and fasting blood glucose levels, alongside decreased insulin secretion and heightened islet cell apoptosis. Moreover, β-cells nuclear condensation and mitochondrial cristae disappearance were observed, accompanied by reduced expression levels of p-GSK-3β protein in islet cells (p < 0.05 or p < 0.01). Conversely, treatment groups administered SQP and Rg displayed augmented expressions of the aforementioned protein markers (p < 0.05 or p < 0.01), alongside preserved mitochondrial cristae structure in islet β cells. Our findings suggest that SQP can ameliorate diabetes by reducing islet cell apoptosis and resist oxidative stress. These insulin-mediated PI3K/AKT/GSK-3β pathway plays an important regulatory role in this process.

Integration of C3H15-mediated transcriptional and post-transcriptional regulation confers plant thermotolerance in Arabidopsis.

Heat stress is an environmental factor that significantly threatens crop production worldwide. Nevertheless, the molecular mechanisms governing plant responses to heat stress are not fully understood. Plant zinc finger CCCH proteins have roles in stress responses as well as growth and development through protein-RNA, protein-DNA, and protein-protein interactions. Here, we reveal an integrated multi-level regulation of plant thermotolerance that is mediated by the CCCH protein C3H15 in Arabidopsis. Heat stress rapidly suppressed C3H15 transcription, which attenuated C3H15-inhibited expression of its target gene HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2), a central regulator of heat stress response (HSR), thereby activating HEAT SHOCK COGNATE 70 (HSC70.3) expression. The RING-type E3 ligase MED25-BINDING RING-H2 PROTEIN 2 (MBR2) was identified as an interacting partner of C3H15. The mbr2 mutant was susceptible to heat stress compared to wild-type plants, whereas plants overexpressing MBR2 showed increased heat tolerance. MBR2-dependent ubiquitination mediated the degradation of phosphorylated C3H15 protein in the cytoplasm, which was enhanced by heat stress. Consistently, heat sensitivities of C3H15 overexpression lines increased in MBR2 loss-of-function and decreased in MBR2 overexpression backgrounds. Heat stress-induced accumulation of HSC70.3 promoted MBR2-mediated degradation of C3H15 protein, implying that an auto-regulatory loop involving C3H15, HSFA2, and HSC70.3 regulates HSR. Heat stress also led to the accumulation of C3H15 in stress granules (SGs), a kind of cytoplasmic RNA granule. This study advances our understanding of the mechanisms plants use to respond to heat stress, which will facilitate technologies to improve thermotolerance in crops.

Presumed hemocytic neoplasms in scorpions

Although neoplasia has been documented in invertebrates, it has not been reported in scorpions. This report describes presumed hemocytic neoplasia in 2 scorpions: a &gt;3-year-old, female emperor scorpion ( Pandinus imperator) and a &gt;4-year-old, male, Asian forest scorpion ( Heterometrus sp.). The emperor scorpion had a 1-month history of body wall swelling separating the exoskeleton of the caudal opisthosoma. At necropsy, this corresponded to a white mass in the caudal coelom. The forest scorpion was found dead and processed whole for histology, at which point multiple masses were identified in the coelom and invading skeletal muscle. Histologically, both masses were composed of sheets of hemocytes with round to oval nuclei; eosinophilic, periodic acid Schiff-positive, cytoplasmic granules; mild cellular atypia; and low mitotic rates. Features of inflammation (e.g., melanization and nodulation) were not observed. These masses were diagnosed as a hemocytoma (emperor scorpion) and a hemocytic sarcoma (forest scorpion), possibly of plasmatocyte origin.

Exploring liquid-liquid phase separation-related diagnostic biomarkers in osteoarthritis based on machine learning algorithms and experiment

BackgroundOsteoarthritis (OA) is a prevalent joint disorder characterized by cartilage degeneration and joint inflammation. Liquid-liquid phase separation (LLPS), a biophysical process involved in cellular organization, has recently gained attention in OA research. However, the relationship between LLPS and OA remains poorly understood. MethodsWe analyzed gene expression data from the GSE48556 dataset to identify LLPS-related genes associated with OA. Differential expression analysis, enrichment analyses, and machine learning algorithms were employed to explore the functional significance of LLPS-related genes in OA and then construct a diagnostic model for OA. In addition, IL-1β as a pro-inflammatory factor to establish an in vitro OA model, and the protein expression levels of OA biomarkers were detected by western blot. ResultsA total of 145 LLPS-related genes were screened in OA samples. Enrichment analyses revealed these genes were mainly enriched in mRNA metabolic processes, cytoplasmic granules, and insulin resistance. Four characteristic genes for OA were selected by using machine learning algorithms, including ADRB2, H3F3B, GNL3L, and PELO. These genes showed satisfactory diagnostic values. Furthermore, there were association between these biomarkers and immune cells, including T cells CD8 and monocytes. In vitro experiments showed that IL-1β stimulation significantly inhibited the cell viability of chondrocytes and enhanced the levels of pro-inflammatory factors, that could mimic the inflammatory state of OA. The expression levels of GNL3L and H3F3B proteins in IL-1β group were obviously lower than those in control group, while levels of ADRB2 and PELO were higher, which was consistent with the results of bioinformatics analysis. ConclusionOur study identifies LLPS-related genes as potential diagnostic biomarkers for OA. These findings provide insights into the molecular mechanisms underlying OA pathogenesis and offer opportunities for the development of novel therapeutic strategies.