This study was aimed at investigating the effectiveness of various host nucleic acid removal and non-specific amplifica-tion techniques in animal tissue samples,to increase the accuracy of pathogen identification in tissue samples.Simulated samples were prepared with a mixture of mouse lung tissue homogenates and Klebsiella pneumoniae fluids,and processed with six host nucleic acid removal kits and three non-specific amplification techniques.The effectiveness of each method in removing host DNA and enriching nucleic acids of pathogenic microorganisms was evaluated through real-time fluorescence quantitative PCR and high-throughput se-quencing.For host nucleic acid removal techniques,the method of selective cleavage and quantitative degradation of host DNA(Com-plete5 kit)effectively decreased the host nucleic acid content in tissue samples and increased the relative abundance of pathogen nucleic acids.In contrast,the magnetic bead method for host DNA removal(Next microbiome DNA enrichment Kit kit)was less effec-tive.At lower pathogen concentrations(77 CFU/mL),the Vazyme kit was more effective than the other kits in removing host nucleic acids.Non-specific amplification techniques(MALBAC whole genome amplification,MDA isothermal amplification,and random primer amplification)were not applicable to tissue samples and were not effective in increasing the relative abundance of pathogen nucleic acids.Selective lysis and quantitative degradation of host DNA were suitable for processing tissue samples with high host back-ground and low pathogenic microorganism levels,whereas non-specific amplification methods were not applicable to tissue samples for pre-processing of macro-genome high-throughput sequencing.

BACKGROUND:Mitochond rial dysfunction leads to cellular senescence and apoptosis,exacerbating tissue damage.Intercellular mitochondrial transfer in injured cells restores mitochondrial function,offering potential therapeutic strategies for mitochondria-related diseases.OBJECTIVE:To review the effects and regulatory mechanisms of intercellular mitochondrial transfer.METHODS:A comprehensive literature search was conducted on mitochondrial transfer between cells in the CNKI and PubMed databases from 2014 to 2024.The Chinese and English search terms used were"mitochondrial transfer,tunneling nanotubes,gap junctions,microvesicles,cell fusion."Eventually,a total of 74 articles were analyzed.RESULTS AND CONCLUSION:(1)The present review provides a comprehensive overview of the four principal mechanisms underlying mitochondrial transfer between cells,encompassing tunneling nanotubes,gap junctions,cell fusion,and microvesicles.(2)This article provides a comprehensive analysis of the pivotal roles played by intercellular mitochondrial transfer,encompassing material exchange,transmission of information,enhancement of host cell mitochondrial function,attenuation of oxidative stress,augmentation of cellular proliferation activity,anti-inflammatory effects,and anti-aging properties.(3)The article provides a comprehensive overview of the main regulatory mechanisms involved in cell mitochondria transfer.These include the promotion of tunneling nanotube formation and mitochondrial transfer by Miro 1,dependence of tunneling nanotubes-mediated mitochondrial transfer on host cell cyclic ADP ribose hydrolase expression,induction of tunneling nanotube formation in an oxidative stress environment,Ca2+-dependent gap junctions,influence of Cx43 on gap junction formation,contribution of Ras1 and actin activation to cell fusion,and involvement of actin and Rab6 in the regulation of mitochondrial exocytosis,activation of actin and NAD+-CD38-cADPR-Ca2+signaling pathways for promoting mitochondrial entry.(4)The transfer of mitochondria occurs via tunneling nanotubes,gap junctions,microvesicles,and cell fusion under the influence of cell signaling proteins,proteins associated with cellular dynamics,and oxidative stress.(5)Mitochondrial transfer plays a pivotal role in facilitating both material and information exchange between cells,thereby intimately linking to the onset and progression of diseases,which can provide new ideas for the treatment of mitochondria-related diseases.However,further investigations are warranted to unravel the effects and regulatory mechanisms of intercellular mitochondrial transfer.

Objective:To evaluate the role of a single PET-CT scan in predicting survival and prognosis in patients with non-small cell lung cancer (NSCLC) who did not undergo surgery but received radiotherapy after neoadjuvant chemotherapy combined with immunotherapy.Methods:A retrospective analysis was conducted on the data of 23 NSCLC patients treated at the Cancer Hospital of the Chinese Academy of Medical Sciences from May 2022 to June 2024. All patients were pathologically confirmed, received neoadjuvant chemotherapy combined with immunotherapy, did not undergo surgery for various reasons, and instead received radiotherapy. Each patient underwent only one PET-CT scan after neoadjuvant chemotherapy combined with immunotherapy and before radiotherapy. According to the maximum standardized uptake value (SUV max) on PET-CT, patients were divided into the low-uptake group (SUV max < 8, n=12) and high-uptake group (SUV max ≥ 8, n=11). Survival analysis was performed using the Kaplan-Meier method with survival curves plotted. Univariate analysis of influencing factors of survival was conducted using the Cox proportional hazards regression model. Clinical characteristics and survival outcomes of the two groups were compared, including progression-free survival (PFS) and overall survival (OS). Results:The 1-year PFS rates were 100% in the low-uptake group, 54.5% in the high-uptake group. This difference was statistically significant ( P=0.007). The 1-year and 2-year OS rates were both 100% in the low-uptake group, the 1-year and 2-year OS rates were both 90.9% in the high-uptake group, with no statistically significant difference ( P=0.394). Univariate Cox analysis identified age as an independent factor affecting PFS. Conclusions:For NSCLC patients who did not undergo surgical resection but received radiotherapy after neoadjuvant chemotherapy combined with immunotherapy, a single PET-CT scan before radiotherapy has potential value in predicting PFS. However, clinical studies with larger sample size and longer follow-up are required to evaluate its predictive value for OS.

BACKGROUND:Mitochond rial dysfunction leads to cellular senescence and apoptosis,exacerbating tissue damage.Intercellular mitochondrial transfer in injured cells restores mitochondrial function,offering potential therapeutic strategies for mitochondria-related diseases.OBJECTIVE:To review the effects and regulatory mechanisms of intercellular mitochondrial transfer.METHODS:A comprehensive literature search was conducted on mitochondrial transfer between cells in the CNKI and PubMed databases from 2014 to 2024.The Chinese and English search terms used were"mitochondrial transfer,tunneling nanotubes,gap junctions,microvesicles,cell fusion."Eventually,a total of 74 articles were analyzed.RESULTS AND CONCLUSION:(1)The present review provides a comprehensive overview of the four principal mechanisms underlying mitochondrial transfer between cells,encompassing tunneling nanotubes,gap junctions,cell fusion,and microvesicles.(2)This article provides a comprehensive analysis of the pivotal roles played by intercellular mitochondrial transfer,encompassing material exchange,transmission of information,enhancement of host cell mitochondrial function,attenuation of oxidative stress,augmentation of cellular proliferation activity,anti-inflammatory effects,and anti-aging properties.(3)The article provides a comprehensive overview of the main regulatory mechanisms involved in cell mitochondria transfer.These include the promotion of tunneling nanotube formation and mitochondrial transfer by Miro 1,dependence of tunneling nanotubes-mediated mitochondrial transfer on host cell cyclic ADP ribose hydrolase expression,induction of tunneling nanotube formation in an oxidative stress environment,Ca2+-dependent gap junctions,influence of Cx43 on gap junction formation,contribution of Ras1 and actin activation to cell fusion,and involvement of actin and Rab6 in the regulation of mitochondrial exocytosis,activation of actin and NAD+-CD38-cADPR-Ca2+signaling pathways for promoting mitochondrial entry.(4)The transfer of mitochondria occurs via tunneling nanotubes,gap junctions,microvesicles,and cell fusion under the influence of cell signaling proteins,proteins associated with cellular dynamics,and oxidative stress.(5)Mitochondrial transfer plays a pivotal role in facilitating both material and information exchange between cells,thereby intimately linking to the onset and progression of diseases,which can provide new ideas for the treatment of mitochondria-related diseases.However,further investigations are warranted to unravel the effects and regulatory mechanisms of intercellular mitochondrial transfer.

This study was aimed at investigating the effectiveness of various host nucleic acid removal and non-specific amplifica-tion techniques in animal tissue samples,to increase the accuracy of pathogen identification in tissue samples.Simulated samples were prepared with a mixture of mouse lung tissue homogenates and Klebsiella pneumoniae fluids,and processed with six host nucleic acid removal kits and three non-specific amplification techniques.The effectiveness of each method in removing host DNA and enriching nucleic acids of pathogenic microorganisms was evaluated through real-time fluorescence quantitative PCR and high-throughput se-quencing.For host nucleic acid removal techniques,the method of selective cleavage and quantitative degradation of host DNA(Com-plete5 kit)effectively decreased the host nucleic acid content in tissue samples and increased the relative abundance of pathogen nucleic acids.In contrast,the magnetic bead method for host DNA removal(Next microbiome DNA enrichment Kit kit)was less effec-tive.At lower pathogen concentrations(77 CFU/mL),the Vazyme kit was more effective than the other kits in removing host nucleic acids.Non-specific amplification techniques(MALBAC whole genome amplification,MDA isothermal amplification,and random primer amplification)were not applicable to tissue samples and were not effective in increasing the relative abundance of pathogen nucleic acids.Selective lysis and quantitative degradation of host DNA were suitable for processing tissue samples with high host back-ground and low pathogenic microorganism levels,whereas non-specific amplification methods were not applicable to tissue samples for pre-processing of macro-genome high-throughput sequencing.

Objective:To evaluate the role of a single PET-CT scan in predicting survival and prognosis in patients with non-small cell lung cancer (NSCLC) who did not undergo surgery but received radiotherapy after neoadjuvant chemotherapy combined with immunotherapy.Methods:A retrospective analysis was conducted on the data of 23 NSCLC patients treated at the Cancer Hospital of the Chinese Academy of Medical Sciences from May 2022 to June 2024. All patients were pathologically confirmed, received neoadjuvant chemotherapy combined with immunotherapy, did not undergo surgery for various reasons, and instead received radiotherapy. Each patient underwent only one PET-CT scan after neoadjuvant chemotherapy combined with immunotherapy and before radiotherapy. According to the maximum standardized uptake value (SUV max) on PET-CT, patients were divided into the low-uptake group (SUV max < 8, n=12) and high-uptake group (SUV max ≥ 8, n=11). Survival analysis was performed using the Kaplan-Meier method with survival curves plotted. Univariate analysis of influencing factors of survival was conducted using the Cox proportional hazards regression model. Clinical characteristics and survival outcomes of the two groups were compared, including progression-free survival (PFS) and overall survival (OS). Results:The 1-year PFS rates were 100% in the low-uptake group, 54.5% in the high-uptake group. This difference was statistically significant ( P=0.007). The 1-year and 2-year OS rates were both 100% in the low-uptake group, the 1-year and 2-year OS rates were both 90.9% in the high-uptake group, with no statistically significant difference ( P=0.394). Univariate Cox analysis identified age as an independent factor affecting PFS. Conclusions:For NSCLC patients who did not undergo surgical resection but received radiotherapy after neoadjuvant chemotherapy combined with immunotherapy, a single PET-CT scan before radiotherapy has potential value in predicting PFS. However, clinical studies with larger sample size and longer follow-up are required to evaluate its predictive value for OS.

Purpose: We aimed to investigate the mechanism of phenotypic transformation of corporal cavernosum smooth muscle cells (CCSMCs) under hypoxic conditions in vitro. Materials and Methods: In this study, a hypoxia model was established using cobalt chloride (CoCl2). CCSMCs were treated with different concentrations of CoCl2 for varying time periods, and cell viability was assessed. Hypoxia-inducible factor-1α (HIF-1α), myocardin (Myocd) and phenotypic markers were detected in the CCSMCs. We also transfected the CCSMCs with si-HIF-1α and Ad-Myocd and evaluated the effects on phenotypic modulation of CCSMCs and the relationship between HIF-1α and Myocd was evaluated. Results: CoCl2 inhibited the viability of CCSMCs in a dose- and time-dependent manner, and treatment with 300 µM CoCl2 for 48 hours were the optimal conditions for establishing the hypoxia model. The results showed increased expression levels of HIF-1α and osteopontin and decreased Myocd, alpha-smooth muscle actin, and calponin levels in CCSMCs under hypoxia. HIF-1α knockdown reversed hypoxia-induced phenotypic transformation with elevated Myocd expression. Overexpression of Myocd also reversed the effect of hypoxia on the phenotypic switch, but did not affect HIF-1α expression. Conclusions Our findings showed that HIF-1α was involved in the effect of hypoxia induced by CoCl2 on CCSMC phenotypic modulation, and Myocd overexpression could inhibit this process. Thus, Myocd might be a potential therapeutic target for erectile dysfunction under hypoxia or HIF-1α activation.

Objective To explore the role of glucose-regulated protein 78 (GRP78),p38 mitogen-activated protein kinase(p38MAPK) signal pathway in seizure-reduced brain injures and the regulatory effect of Nimodipine on it.Methods Sprague-Dawley(SD) rats were randomly divided into status convulsion group (SC group),Nimodipine group(NM group),and a normal control group(NC group).The expressions of GRP78/Bip and p38MAPK mRNA and protein were detected by reverse transcription(RT)-PCR and immunohistochemistry.The expression of apoptosis cells was observed by TdT-mediated dUTP nick end labeling (TUNEL).Results (1) Immunohistochemistry:at 4 h after induction of status convulsion,the expression of GRP78 protein in the hippocampus CA1 domain began increasing slightly,and reached a maximum at 24 h,and then began decreasing slowly ; at 4 h after induction of status convulsion,the expression of p38MAPK protein in the hippocampus CA1 domain began increasing,and reached a maximum at 24 h,and decreased remarkably at 48 h.(2) RT-PCR:at 4 h after induction of status convulsion,the expression of GRP78 protein in the hippocampus CA1 domain began increasing slightly,and reached a maximum at 24 h,and then began decreasing slowly.The NM group was much higher than the SC group and the NC group(all P ＜ 0.05) ; at 4 h after induction of status convulsion,the expression of p38MAPK protein in the hippocampus CA1 domain began increasing,and reached a maximum at 24 h,and decreased remarkably at 48 h;the NM group was much lower than the SC group,and higher than the NC group (all P ＜ 0.05).(3) TUNEL:at 4 h after induction of status convulsion,the expression of the TUNEL positive cells in the hippocampus CA1 domain began increasing slightly,and reached a maximum at 48 h,and then began decreasing,and there was no difference between SC group and NC group;the NM group was much lower than the SC group(all P ＜ 0.05).Conclusions The correlation of the increased expression of p38MAPK and neuronal apoptosis indicates that GRP78 signal pathway may be mediated to cell apoptosis through p38MAPK.Nimodipine can affect the expression of GRP78/Bip and p38MAPK,and relieve endoplasmic reticulum stress,and lessen the pathologic damage to the hippocampus.

[ABSTRACT]AIM:Tostudytheeffectsofbaicalin(BC)onglialfibrillaryacidicprotein(GFAP)andnuclear factor-κB ( NF-κB) expression and neuronal apoptosis in juvenile rat hippocampus after status convulsion ( SC) .METH-ODS:One hundred and ninety five juvenile male Sprague-Dawley rats were randomly divided into 3 groups:normal saline pretreatment group (NS group), SC group and SC with BC pretreatment group (BC group).Each of these 3 groups would be subdivided into 5 subgroups sacrificed at 4 h, 12 h, 24 h, 48 h and 72 h after SC.The rat SC model was prepared by lithium-pilocarpine chemical method .The protein expression of GFAP and NF-κB was detected by the method of immuno-histochemistry .The mRNA expression of GFAP was detected by RT-PCR.The neuronal apoptosis was observed by TdT-mediated dUTP nick end labeling ( TUNEL ) .RESULTS: Compared with NS group , the GFAP positive cells was in-creased in SC group (P<0.05).Compared with SC group, the expression of GFAP was significantly reduced in BC group (P<0.05).Compared with NS group, the NF-κB positive cells was increased in SC group (P<0.05).Compared with SC group, the expression of NF-κB was significantly reduced in BC group .RT-PCR showed that the expression trend of GFAP mRNA was similar to that of the protein .Compared with NS group , the TUNEL positive cells in the hippocampus CA1 area in SC group increased significantly 12 h after SC (P<0.01), and reached a peak at 48 h.After the intervention with BC, the TUNEL positive cells decreased significantly between 12~48 h after SC (P<0.05 or P<0.01), but the number of TUNEL positive cells remained significantly greater than that in NS group (P<0.05).CONCLUSION: The expression of GFAP and NF-κB in the hippocampus increased after SC in rats .Baicalin decreases the expression of GFAP and NF-κB in hippocampus of rats with pilocarpine-induced seizures , and reduces the number of neuronal apoptosis , sug-gesting that baicalin may protect against the brain damage caused by status convulsion .

Objective To study the expression of toll-like receptor 4 (TLR4), nuclear factor κB (NF-кB) and Caspase-3 and the change of neuron apoptosis of the hippocampus in the status convulsion rat , and to explore the effect of resveratrol on them.Methods A total of 106 male Sprague-Dawley(SD)rats were randomly divided into the control (A), convulsion (B) and resveratrol treatment (C) groups.Group B was further randomly divided into four subset groups (B1-B4) which were executed at 4h, 24h, 48h, 72h after convulsion discontinued .Continuous epilepticus was induced by injecting lithium chloride and pilocarpine , and group C was daily injected with 30mg/kg resveratrol 30minutes after convulsion stopped for 3 days.TLR4 and NF-κB/p65 proteins were detected by immunohistochemistry ( IHC ); the expressions of TLR4 and Caspase-3 mRNA were detected by RT-PCR.The neuron apoptosis was observed by TUNEL . Results The IHC staining of TLR4 protein in group B was significantly higher than in group A (P<0.05), and that in group C was much lower than in B 4 group ( P<0.01 ) .The expression of NF-кB/p65 showed that hippocampal neurons had positive expression in cell nuclei in group B compared with group A (P<0.05), and the expression of NF-кB/p65 protein in group C was much lower than that in group B 4 (P<0.01).The mRNA expressions of TLR4 and Caspase-3 in the rat hippocampus of group B were significantly elevated than that in group A ( P <0.05 ) , and the tendency was increased gradually , reaching the peak at 72 hours after seizure , and that in group C was much lower than that in B 4 group (P<0.01).The TUNEL positive cells in hippocampus CAl of B group were more than that of group A after the SC 24hours (P<0.01),reached the highest level at the 72nd hour, and that in group C the TUNEL positive cells were lower than that in B4 group (P<0.01).Conclusion The expression of TLR4, NF-кB and Caspase-3 increased after SC.Resveratrol can down regulate the expression of TLR4, NF-кB and Caspase-3 in the hippocampus with pilocarpine-induced seizures, reduced the number of neuronal apoptosis .These results suggest that resveratrol may have a protective effect against the hippocampus damage caused by status convulsion .