ObjectiveThe malaria parasites remodel the host erythrocyte structure by exporting parasite proteins that interact with the membrane skeleton proteins of red blood cells (RBCs), facilitating their intracellular survival and pathogenicity. Skeleton-binding protein 1 (SBP1) is a conserved exported protein across Plasmodium species. In Plasmodium falciparum, SBP1 has been reported to interact with erythrocyte membrane skeleton proteins 4.1R and spectrin, while its contribution to erythrocyte remodeling and parasite virulence in Plasmodium berghei (Pb) remains unclear. This study aims to determine whether PbSBP1 associates with the host cytoskeletal protein 4.1R and to investigate its role in the remodeling of host RBCs and the pathogenicity of Plasmodium berghei. MethodsIn Plasmodium berghei, the relationship between PbSBP1 and the erythrocyte cytoskeletal protein 4.1R was examined using co-immunoprecipitation. A Pbsbp1 gene knockout mutant of Plasmodium berghei (Pbsbp1∆) was generated based on the principle of double crossover homologous recombination. The deformability of erythrocytes infected with Pbsbp1∆ parasites was assessed using microfluidic methods. Microchannels with an array of cylindrical pillars were used to detect modifications in infected RBC deformability. The infected RBCs were squashed between the rows and recovered between the columns and the transit velocity (μm/s) of infected RBCs travelling through the microchannel was recorded. The component of the erythrocyte membrane skeleton junctional complex, tropomodulin (TMOD), was fluorescently labeled, and the cytoskeletal network of infected erythrocytes was imaged using super-resolution stochastic optical reconstruction microscopy (STORM) to analyze ultrastructural changes in the cytoskeleton of wild-type (WT) and Pbsbp1∆-infected erythrocytes. Actin-based junctional complexes were displayed as individual clusters by the labeled TMOD in the STORM images, and the cluster densities and distances between adjacent clusters of infected RBCs were calculated. Additionally, rodent malaria models (BALB/c mice) and experimental cerebral malaria models (C57BL/6 mice) were employed to monitor the growth of Pbsbp1∆ and WT parasites during the intraerythrocytic stage and their capacity to induce cerebral malaria in mice. ResultsPbSBP1 may participate in the remodeling of infected erythrocytes through direct or indirect interaction with the erythrocyte cytoskeletal protein 4.1R. Microfluidic assays revealed that the deformability of erythrocytes infected with Pbsbp1∆ parasites was significantly enhanced compared to those infected with WT parasites. STORM imaging further demonstrated that the ultrastructure of the erythrocyte cytoskeleton in Pbsbp1∆-infected cells was altered relative to that in WT-infected erythrocytes. The distances between nearest neighbors of clusters had a tendency to increase while the cluster densities were decreased in Pbsbp1∆-infected RBCs compared to WT-infected RBCs. Subsequent phenotypic analysis indicated that the growth rate of Pbsbp1∆ parasites during the intraerythrocytic stage was significantly slower than that of WT parasites, and their ability to induce cerebral malaria in mice was also attenuated. These findings suggest that PbSBP1 is involved in the remodeling of the erythrocyte membrane skeleton, likely through its direct or indirect interaction with protein 4.1R, thereby regulating the deformability of infected erythrocytes and influencing the pathogenicity of the blood-stage parasites. ConclusionThis study establishes a role for PbSBP1 in host erythrocyte remodeling and parasite virulence, providing new research strategies for the prevention and treatment of malaria.

ObjectiveTo explore effect of Xianlian Jiedu prescription on the recurrence of colorectal cancer （CRC） and investigate the related mechanisms. MethodsA postoperative recurrence model was established in 25 Balb/c mice by injecting CT26 cells subcutaneously into the armpit， followed by surgical removal of 99% of the subcutaneous tumor. The mice were randomly divided into model group， low-dose Xianlian Jiedu prescription （XLJDP-L） group （6.45 g·kg^-1·d^-1）， medium-dose Xianlian Jiedu prescription （XLJDP-M） group （12.9 g·kg^-1·d^-1）， high-dose Xianlian Jiedu prescription （XLJDP-H） group （25.8 g·kg^-1·d^-1）， and 5-fluorouracil （5-FU） group （1×10^-3 g·kg^-1·d^-1）. The mice were euthanized after 14 days of continuous intervention， and recurrent tumor tissue was harvested. Hematoxylin and eosin （HE） staining was used to observe pathological and morphological changes in the recurrent tumor tissue. Immunohistochemistry （IHC） was employed to assess the expression of proliferating cell nuclear antigen （Ki67）， vascular endothelial growth factor （VEGF）， and platelet-endothelial cell adhesion molecule （CD31） in recurrent tumor tissue. The Western blot was used to detect the protein expression levels of angiopoietin-2 （ANG-2）， VEGF， phosphorylated-protein kinase B （p-Akt）， protein kinase B （Akt）， phosphorylated-phosphatidylinositol 3-kinase （p-PI3K）， and phosphatidylinositol 3-kinase （PI3K） in recurrent tumor tissue. ResultsBefore treatment， there were no statistical differences in tumor volume， tumor weight， and body mass among the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group compared to the model group， indicating model stability. After treatment， compared with those in the model group， the tumor volume and tumor weight in the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group were significantly reduced （P<0.01）， showing dose dependency. Meanwhile， there were no significant differences in body weight among the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group compared to the model group. HE staining showed that compared with that in the model group， tumor tissue in the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group had loosely arranged cells， increased intercellular spaces， small and shriveled nuclei， light staining， fewer mitotic figures and atypical nuclei， and increased necrotic areas. IHC showed that compared with those of the model group， the positive rates of Ki67， VEGF， and CD31 in the recurrent tumor tissue of the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group were significantly reduced （P<0.01） in a dose-dependent manner. Western blot results showed that compared with those of the model group， the protein expression levels of ANG-2 and VEGF in the recurrent tumor tissue of the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group were significantly downregulated （P<0.05， P<0.01）， and the p-Akt/Akt and p-PI3K/PI3K ratios were significantly decreased in a dose-dependent manner （P<0.05， P<0.01）. ConclusionXianlian Jiedu prescription significantly inhibits the recurrence of CRC in mice after subcutaneous tumor surgery. The mechanism may involve regulating the PI3K/Akt pathway and downregulating key angiogenic proteins such as ANG-2， VEGF， and CD31.

Subarachnoid hemorrhage (SAH) is a serious intracranial hemorrhage characterized by acute bleeding into the subarachnoid space. The effects of shikonin, a natural compound from the roots of Lithospermum erythrorhizon, on oxidative stress and blood–brain barrier (BBB) injury in SAH was evaluated in this study. A rat model of SAH was established by endovascular perforation to mimic the rupture of intracranial aneurysms. Rats were then administered 25 mg/kg of shikonin or dimethylsulfoxide after surgery. Brain edema, SAH grade, and neurobehavioral scores were measured after 24 h of SAH to evaluate neurological impairment. Concentrations of the oxidative stress markers superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) in the brain cortex were determined using the corresponding commercially available assay kits. Evans blue staining was used to determine BBB permeability. Western blotting was used to quantify protein levels of tight junction proteins zonula occludens-1, Occludin, and Claudin-5. After modeling, the brain water content increased significantly whereas the neurobehavioral scores of rats with SAH decreased prominently. MDA levels increased and the levels of the antioxidant enzymes GSH and SOD decreased after SAH. These changes were reversed after shikonin administration. Shikonin treatment also inhibited Evans blue extravasation after SAH. Furthermore, reduction in the levels of tight junction proteins after SAH modeling was rescued after shikonin treatment. In conclusion, shikonin exerts a neuroprotective effect after SAH by mitigating BBB injury and inhibiting oxidative stress in the cerebral cortex.

Subarachnoid hemorrhage (SAH) is a serious intracranial hemorrhage characterized by acute bleeding into the subarachnoid space. The effects of shikonin, a natural compound from the roots of Lithospermum erythrorhizon, on oxidative stress and blood–brain barrier (BBB) injury in SAH was evaluated in this study. A rat model of SAH was established by endovascular perforation to mimic the rupture of intracranial aneurysms. Rats were then administered 25 mg/kg of shikonin or dimethylsulfoxide after surgery. Brain edema, SAH grade, and neurobehavioral scores were measured after 24 h of SAH to evaluate neurological impairment. Concentrations of the oxidative stress markers superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) in the brain cortex were determined using the corresponding commercially available assay kits. Evans blue staining was used to determine BBB permeability. Western blotting was used to quantify protein levels of tight junction proteins zonula occludens-1, Occludin, and Claudin-5. After modeling, the brain water content increased significantly whereas the neurobehavioral scores of rats with SAH decreased prominently. MDA levels increased and the levels of the antioxidant enzymes GSH and SOD decreased after SAH. These changes were reversed after shikonin administration. Shikonin treatment also inhibited Evans blue extravasation after SAH. Furthermore, reduction in the levels of tight junction proteins after SAH modeling was rescued after shikonin treatment. In conclusion, shikonin exerts a neuroprotective effect after SAH by mitigating BBB injury and inhibiting oxidative stress in the cerebral cortex.

Purpose: This study aimed to investigate the molecular characteristics associated with better prognosis in breast cancer. Methods: We performed targeted sequencing of 962 genes in 56 samples, categorizing them into long-term and short-term survival groups as well as chemotherapy-sensitive and chemotherapy-resistant groups for further analyses. Results: The results indicated that the tumor mutational burden values were significantly higher in the short-term survival and chemotherapy-resistant groups (p = 0.008 and p = 0.003, respectively). Somatic mutation analysis revealed that the mutation frequencies of BCL9L and WHSC1 were significantly lower in the long-term survival group than those in the short-term survival group (p = 0.029 and p = 0.024, respectively). CREB-regulated transcription coactivator 1 (CRTC1) mutations occurred significantly more frequently in the chemotherapy-resistant group (p = 0.027) and were associated with shorter progression-free survival (p = 0.036).Signature weighting analysis showed a significant increase in Signature.3, which is associated with homologous recombination repair deficiency in the chemotherapy-sensitive group (p = 0.045). Conversely, signatures related to effective DNA repair mechanisms, Signature.1 and Signature.15, were significantly reduced (p = 0.002 and p < 0.001, respectively). Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that gene mutations were significantly enriched in the JAK-STAT signaling pathway. Conclusion This study, through intergroup comparative analysis, found that immunotherapy (using programmed death 1/programmed death-ligand 1 inhibitors) may improve the prognosis of patients with short survival and chemotherapy resistance. Additionally, the study revealed that mutations in BCL9L and WHSC1 could serve as biomarkers for breast cancer prognosis, while CRTC1 mutations and Signature.3 could predict chemotherapy response. The study also found that the JAK-STAT pathway might be a potential therapeutic target for chemotherapy resistance. Therefore, this study identifies molecular characteristics that influence the prognosis of breast cancer patients, providing important theoretical insights for the development of personalized treatment strategies.

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

Objective: Huntington’s disease (HD) is characterized by motor, cognitive, and neuropsychiatric symptoms. Oculomotor impairments and gait variability have been independently considered as potential markers in HD. However, an integrated analysis of eye movement and gait is lacking. We performed multiple examinations of eye movement and gait variability in HTT mutation carriers, analyzed the consistency between these parameters and clinical severity, and then examined the associations between oculomotor impairments and gait deficits. Methods: We included 7 patients with pre-HD, 30 patients with HD and 30 age-matched controls. We collected demographic data and assessed the Unified Huntington’s Disease Rating Scale (UHDRS) score. Examinations, including saccades, smooth pursuit tests, and optokinetic (OPK) tests, were performed to evaluate eye movement function. The parameters of gait include stride length, walking velocity, step deviation, step length, and gait phase. Results: HD patients have significant impairments in the latency and velocity of saccades, the gain of smooth pursuit, and the gain and slow phase velocities of OPK tests. Only the speed of saccades significantly differed between pre-HD patients and controls. There are significant impairments in stride length, walking velocity, step length, and gait phase in HD patients. The parameters of eye movement and gait variability in HD patients were consistent with the UHDRS scores. There were significant correlations between eye movement and gait parameters. Conclusion Our results show that eye movement and gait are impaired in HD patients and that the speed of saccades is impaired early in pre-HD. Eye movement and gait abnormalities in HD patients are significantly correlated with clinical disease severity.

Subarachnoid hemorrhage (SAH) is a serious intracranial hemorrhage characterized by acute bleeding into the subarachnoid space. The effects of shikonin, a natural compound from the roots of Lithospermum erythrorhizon, on oxidative stress and blood–brain barrier (BBB) injury in SAH was evaluated in this study. A rat model of SAH was established by endovascular perforation to mimic the rupture of intracranial aneurysms. Rats were then administered 25 mg/kg of shikonin or dimethylsulfoxide after surgery. Brain edema, SAH grade, and neurobehavioral scores were measured after 24 h of SAH to evaluate neurological impairment. Concentrations of the oxidative stress markers superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) in the brain cortex were determined using the corresponding commercially available assay kits. Evans blue staining was used to determine BBB permeability. Western blotting was used to quantify protein levels of tight junction proteins zonula occludens-1, Occludin, and Claudin-5. After modeling, the brain water content increased significantly whereas the neurobehavioral scores of rats with SAH decreased prominently. MDA levels increased and the levels of the antioxidant enzymes GSH and SOD decreased after SAH. These changes were reversed after shikonin administration. Shikonin treatment also inhibited Evans blue extravasation after SAH. Furthermore, reduction in the levels of tight junction proteins after SAH modeling was rescued after shikonin treatment. In conclusion, shikonin exerts a neuroprotective effect after SAH by mitigating BBB injury and inhibiting oxidative stress in the cerebral cortex.

Subarachnoid hemorrhage (SAH) is a serious intracranial hemorrhage characterized by acute bleeding into the subarachnoid space. The effects of shikonin, a natural compound from the roots of Lithospermum erythrorhizon, on oxidative stress and blood–brain barrier (BBB) injury in SAH was evaluated in this study. A rat model of SAH was established by endovascular perforation to mimic the rupture of intracranial aneurysms. Rats were then administered 25 mg/kg of shikonin or dimethylsulfoxide after surgery. Brain edema, SAH grade, and neurobehavioral scores were measured after 24 h of SAH to evaluate neurological impairment. Concentrations of the oxidative stress markers superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) in the brain cortex were determined using the corresponding commercially available assay kits. Evans blue staining was used to determine BBB permeability. Western blotting was used to quantify protein levels of tight junction proteins zonula occludens-1, Occludin, and Claudin-5. After modeling, the brain water content increased significantly whereas the neurobehavioral scores of rats with SAH decreased prominently. MDA levels increased and the levels of the antioxidant enzymes GSH and SOD decreased after SAH. These changes were reversed after shikonin administration. Shikonin treatment also inhibited Evans blue extravasation after SAH. Furthermore, reduction in the levels of tight junction proteins after SAH modeling was rescued after shikonin treatment. In conclusion, shikonin exerts a neuroprotective effect after SAH by mitigating BBB injury and inhibiting oxidative stress in the cerebral cortex.

Objective: Huntington’s disease (HD) is characterized by motor, cognitive, and neuropsychiatric symptoms. Oculomotor impairments and gait variability have been independently considered as potential markers in HD. However, an integrated analysis of eye movement and gait is lacking. We performed multiple examinations of eye movement and gait variability in HTT mutation carriers, analyzed the consistency between these parameters and clinical severity, and then examined the associations between oculomotor impairments and gait deficits. Methods: We included 7 patients with pre-HD, 30 patients with HD and 30 age-matched controls. We collected demographic data and assessed the Unified Huntington’s Disease Rating Scale (UHDRS) score. Examinations, including saccades, smooth pursuit tests, and optokinetic (OPK) tests, were performed to evaluate eye movement function. The parameters of gait include stride length, walking velocity, step deviation, step length, and gait phase. Results: HD patients have significant impairments in the latency and velocity of saccades, the gain of smooth pursuit, and the gain and slow phase velocities of OPK tests. Only the speed of saccades significantly differed between pre-HD patients and controls. There are significant impairments in stride length, walking velocity, step length, and gait phase in HD patients. The parameters of eye movement and gait variability in HD patients were consistent with the UHDRS scores. There were significant correlations between eye movement and gait parameters. Conclusion Our results show that eye movement and gait are impaired in HD patients and that the speed of saccades is impaired early in pre-HD. Eye movement and gait abnormalities in HD patients are significantly correlated with clinical disease severity.