Mitigation of benzyl butyl phthalate toxicity in male germ cells with combined treatment of parthenolide, N-acetylcysteine, and 3-methyladenine.

Benzyl butyl phthalate (BBP) is a widely used plasticizer that poses various potential health hazards. Although BBP has been extensively studied, the direct mechanism underlying its toxicity in male germ cells remains unclear. Therefore, we investigated BBP-mediated male germ cell toxicity in GC-1 spermatogonia (spg), a differentiated mouse male germ cell line. This study investigated the impact of BBP on reactive oxygen species (ROS) generation, apoptosis, and autophagy regulation, as well as potential protective measures against BBP-induced toxicity. A marked dose-dependent decrease in GC-1 spg cell proliferation was observed following treatment with BBP at 12.5 µM. Exposure to 50 µM BBP, approximating the IC50 of 53.9 µM, markedly increased cellular ROS generation and instigated apoptosis, as evidenced by augmented protein levels of both intrinsic and extrinsic apoptosis-related markers. An amount of 50 µM BBP induced marked upregulation of autophagy regulator proteins, p38 MAPK, and extracellular signal-regulated kinase and substantially downregulated the phosphorylation of key kinases involved in regulating cell proliferation, including phosphoinositide 3-kinase, protein kinase B, mammalian target of rapamycin (mTOR), c-Jun N-terminal kinase. The triple combination of N-acetylcysteine, parthenolide, and 3-methyladenine markedly restored cell proliferation, decreased BBP-induced apoptosis and autophagy, and restored mTOR phosphorylation. This study provides new insights into BBP-induced male germ cell toxicity and highlights the therapeutic potential of the triple inhibitors in mitigating BBP toxicity.

Magnet-Guided Temozolomide and Ferucarbotran Loaded Nanoparticles to Enhance Therapeutic Efficacy in Glioma Model.

Background. The aim of the study was to synthesize liposomal nanoparticles loaded with temozolomide and ferucarbotran (LTF) and to evaluate the theranostic effect of LTF in the glioma model. Methods. We synthesized an LTF that could pass through the Blood Brain Barrier (BBB) and localize in brain tumor tissue with the help of magnet guidance. We examined the chemical characteristics. Cellular uptake and cytotoxicity studies were conducted in vitro. A biodistribution and tumor inhibition study was conduted using an in vivo glioma model. Results. The particle size and surface charge of LTF show 108 nm and -38 mV, respectively. Additionally, the presence of ferucarbotran significantly increased the contrast agent effect of glioma compared to the control group in MR imaging. Magnet-guided LTF significantly reduced the tumor size compared to control and other groups. Furthermore, compared to the control group, our results demonstrate a significant inhibition in brain tumor size and an increase in lifespan. Conclusions. These findings suggest that the LTF with magnetic guidance represents a novel approach to address current obstacles, such as BBB penetration of nanoparticles and drug resistance. Magnet-guided LTF is able to enhance therapeutic efficacy in mouse brain glioma.

Ultrasonographic images and correspondence with real color sectioned images of the upper limb.

PURPOSE: For basic training in ultrasonography (US), medical students and residents must learn cross-sectional anatomy. However, the present educational material is not sufficient to learn the sectional anatomy for US. This study aimed to provide a criterion for reading ambiguous structures on US images of upper limb through the sectioned images of Visible Korean. METHODS: US images of the right arm of a volunteer were scanned (28 planes). For comparison with US images, the sectioned images of the right upper limb (24 bits color, 0.5 mm intervals, 0.06 mm × 0.06 mm sized pixel) were used. After the volume model was constructed from the sectioned images using MRIcroGL, new sectioned images of 28 planes corresponding to the US images of 28 planes were created by adjusting the slope of the volume model. In all images, the anatomical terms of 59 structures from the shoulder to the fingers were annotated. RESULTS: In the atlas, which consists of 28 sets of US images and sectioned images of various slope planes, 59 structures of the shoulder, arm, elbow, wrist, palm, and fingers were observed in detail. CONCLUSION: We were able to interpret the ambiguous structures on the US images using the sectioned images with high resolution and actual color. Therefore, to learn the cross-sectional anatomy for US, the sectioned images from the Visible Korean project were deemed to be the suitable data because they contained all human gross anatomical information.

Inhibition of reactive oxygen species generation by N-Acetyl Cysteine can mitigate male germ cell toxicity induced by bisphenol analogs.

The estrogen-like effect of bisphenol A (BPA) disrupting the maintenance of functional male germ cells is associated with male sub-fertility. This study investigated toxicity of male germ cells induced by four bisphenol analogs: BPA, BPAF, BPF, and BPS. The investigation of bisphenol analogs' impact on male germ cells included assessing proliferation, apoptosis induction, and the capacity to generate reactive oxygen species (ROS) in GC-1 spermatogonia (spg) cells, specifically type B spermatogonia. Additionally, the therapeutic potential and protective effects of N-Acetyl Cysteine (NAC) and NF-κB inhibitor parthenolide was evaluated. In comparison to BPA, BPF and BPS, BPAF exhibited the most pronounced adverse effect in GC-1 spg cell proliferation. This effect was characterized by pronounced inhibition of phosphorylation of PI3K, AKT, and mTOR, along with increased release of cytochrome c and subsequent cleavages of caspase 3, caspase 7, and poly (ADP-ribose) polymerase. Both NAC and parthenolide were effective reducing cellular ROS induced by BPAF. However, only NAC demonstrated a substantial recovery in proliferation, accompanied by a significant reduction in cytochrome c release and cleaved PARP. These results suggest that NAC supplementation may play an effective therapeutic role in countering germ cell toxicity induced by environmental pollutants with robust oxidative stress-generating capacity.

Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integration.

High-yield production of therapeutic protein using Chinese hamster ovary (CHO) cells requires stable cell line development (CLD). CLD typically uses random integration of transgenes; however, this results in clonal variation and subsequent laborious clone screening. Therefore, site-specific integration of a protein expression cassette into a desired chromosomal locus showing high transcriptional activity and stability, referred to as a hot spot, is emerging. Although positional effects are important for therapeutic protein expression, the sequence-specific mechanisms by which hotspots work are not well understood. In this study, we performed whole-genome sequencing (WGS) to locate randomly inserted vectors in the genome of recombinant CHO cells expressing high levels of monoclonal antibodies (mAbs) and experimentally validated these locations and vector compositions. The integration site was characterized by active histone marks and potential enhancer activities, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated indel mutations in the region upstream of the integration site led to a significant reduction in specific antibody productivity by up to 30%. Notably, the integration site and its core region did not function equivalently outside the native genomic context, showing a minimal effect on the increase in exogenous protein expression in the host cell line. We also observed a superior production capacity of the mAb expressing cell line compared to that of the host cell line. Collectively, this study demonstrates that developing recombinant CHO cell lines to produce therapeutic proteins at high levels requires a balance of factors including transgene configuration, genomic locus landscape, and host cell properties.

Antioxidant and Anti-Fatigue Effects of a Standardized Botanical Extract Fraction (HemoHIM) in Forced-Exercised Aged Mice.

HemoHIM is a standardized medicinal herbal preparation consisting of extracts of Angelica gigas Nakai, Cnidium officinale Makino, and Paeonia lactiflora Pallas that possesses immune regulatory activities. This study aimed to research the potential antioxidant effects of HemoHIM and its capacity for reducing fatigue in aged mice subjected to forced exercise. After administering HemoHIM 125 (500 mg/kg orally) for 4 weeks in 8-month-old female C57BL/6 mice (4 groups of 10 mice), various parameters were evaluated. The analyses revealed that HemoHIM enhanced swimming time and grip strength. In addition, it significantly reduced serum lactate levels and increased liver glutathione peroxidase (GPx) levels after exercise challenge. The expression levels of antioxidant enzymes and factors, including nuclear factor erythroid 2-related factor-2 (Nrf-2), heme oxygenase 1, superoxide dismutase, GPx, and glutathione reductase, were significantly higher in liver and muscle tissues of mice treated with HemoHIM. These results indicate that HemoHIM might function as an anti-fatigue and antioxidant agent by modulating the Nrf-2 signaling pathway.

Impact of Coronavirus Disease 2019 on Unresectable Hepatocellular Carcinoma Treated with Atezolizumab/Bevacizumab.

(1) Background: The coronavirus disease 2019 (COVID-19) pandemic has proven challenging to the management of patients with cancer, particularly those receiving systemic therapy. This study aimed to evaluate the impact of COVID-19 on patients with unresectable hepatocellular carcinoma (HCC) treated with atezolizumab/bevacizumab. (2) Methods: Patients with unresectable HCC who started atezolizumab/bevacizumab treatment between June 2020 and December 2021 at a tertiary cancer center in Korea were included (n = 241) and classified according to their COVID-19 status and severity. (3) Results: Thirty-five (14.5%) patients with unresectable HCC were diagnosed with COVID-19 during atezolizumab/bevacizumab treatment; 26 (74.2%) and nine (25.7%) in the low- and high-severity groups, respectively. The high-severity group showed higher neutrophil-to-lymphocyte ratios and lactate dehydrogenase levels. Liver and kidney injuries were observed in 31.4% and 17.1% of total patients, respectively. Liver injury was more prominent in patients with pre-existing liver dysfunction at baseline, who were more prevalent in the high-severity group. Atezolizumab/bevacizumab treatment was delayed by a median of 0 (range, 0-21) day in the low-severity group and 12 (range, 0-35) days in the high-severity group. The high-severity group showed worse post-infection progression-free survival (1.1 vs. 4.8 months, p = 0.017) and overall survival (2.2 months vs. not reached, p = 0.004). (4) Conclusions: Patients with impaired liver function at baseline are more susceptible to high-severity COVID-19, which affects atezolizumab/bevacizumab treatment outcomes.

Surgical and Fertility Outcomes of Reduced-Port Robotic Myomectomy: A Single-Center Experience of 401 Cases.

Background: Reduced-port robotic myomectomy (RPRM) using Da Vinci® Xi™ is a good fertility-saving surgical option, but the surgical and fertility outcomes are unknown. Methods: This was a retrospective cohort study evaluating the feasibility of RPRM in an academic tertiary hospital setting. A total of 401 patients who underwent RPRM by a single operator between October 2017 and October 2021 were included. For RPRM, three ports are required: a 1.5 cm umbilical incision and two 0.8 cm incisions 8 cm lateral to the umbilicus. A single-port system was applied through the umbilicus, which also functioned as a working port. Unlike conventional robotic surgery, only three robot arms were utilized for the entire surgical procedure. Results: Surgical and fertility outcomes were assessed through medical records review and follow-up telephone contact. The mean age of patients at the time of surgery was 39.7 ± 6.0 years. The most common indication for surgery was menorrhagia (n = 128, 31.9%). The average number of myomas removed was 4.7 ± 4.1 (1-22), and the size was 7.8 ± 2.5 cm (2.5-16.0). The mean operation time was 103.7 ± 45.6 min. Postoperative complications were found in 9.7% (n = 39) of patients; the most common complication was transfusion (7.7%, n = 31). After surgery, 70 patients tried to conceive, and 56 became pregnant naturally or by assisted reproduction (56/70, 80.0%). The mean interval time from operation to conception was 13.5 ± 10.1 months. Among 56 who conceived, 44 gave birth (62.9%), five were still ongoing (7.1%), and seven had miscarriages (10.0%). Cesarean section was performed for most cases (43/44, 97.7%). Eight patients had obstetric complications (16.3%), but no uterine rupture was reported. Conclusions: RPRM, which provides the benefits of conventional robotic surgery along with favorable obstetric and cosmetic results, is a feasible option for patients with symptomatic uterine myomas who wish to conceive in the future.

Electroencephalography-based classification of Alzheimer's disease spectrum during computer-based cognitive testing.

Alzheimer's disease (AD) is a progressive disease leading to cognitive decline, and to prevent it, researchers seek to diagnose mild cognitive impairment (MCI) early. Particularly, non-amnestic MCI (naMCI) is often mistaken for normal aging as the representative symptom of AD, memory decline, is absent. Subjective cognitive decline (SCD), an intermediate step between normal aging and MCI, is crucial for prediction or early detection of MCI, which determines the presence of AD spectrum pathology. We developed a computer-based cognitive task to classify the presence or absence of AD pathology and stage within the AD spectrum, and attempted to perform multi-stage classification through electroencephalography (EEG) during resting and memory encoding state. The resting and memory-encoding states of 58 patients (20 with SCD, 10 with naMCI, 18 with aMCI, and 10 with AD) were measured and classified into four groups. We extracted features that could reflect the phase, spectral, and temporal characteristics of the resting and memory-encoding states. For the classification, we compared nine machine learning models and three deep learning models using Leave-one-subject-out strategy. Significant correlations were found between the existing neurophysiological test scores and performance of our computer-based cognitive task for all cognitive domains. In all models used, the memory-encoding states realized a higher classification performance than resting states. The best model for the 4-class classification was cKNN. The highest accuracy using resting state data was 67.24%, while it was 93.10% using memory encoding state data. This study involving participants with SCD, naMCI, aMCI, and AD focused on early Alzheimer's diagnosis. The research used EEG data during resting and memory encoding states to classify these groups, demonstrating the significance of cognitive process-related brain waves for diagnosis. The computer-based cognitive task introduced in the study offers a time-efficient alternative to traditional neuropsychological tests, showing a strong correlation with their results and serving as a valuable tool to assess cognitive impairment with reduced bias.

Characteristics of the Discoloration Switching Phenomenon of 4H-SiC Single Crystals Grown by PVT Method Using ToF-SIMS and Micro-Raman Analysis.

The discoloration switching appearing in the initial and final growth stages of 4H-silicon carbide (4H-SiC) single crystals grown using the physical vapor transport (PVT) technique was investigated. This phenomenon was studied, investigating the correlation with linear-type micro-pipe defects on the surface of 4H-SiC single crystals. Based on the experimental results obtained using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and micro-Raman analysis, it was deduced that the orientation of the 4H-SiC c-axis causes an axial change that correlates with low levels of carbon. In addition, it was confirmed that the incorporation of additional elements and the concentrations of these doped impurity elements were the main causes of discoloration and changes in growth orientation. Overall, this work provides guidelines for evaluating the discoloration switching in 4H-SiC single crystals and contributes to a greater understanding of this phenomenon.

An 8-mm trocar-site hernia at a drainage insertion site after a three-port robotic myomectomy: case report and review of literature.

Trocar site hernia is a rare, serious operation-related complication after robotic gynecologic surgery. Here, we present two 8-mm port-site hernia cases after three-port robotic myomectomy with a review of reported previous cases. In the first case, small bowel obstruction was found postoperatively due to herniation at the left mid-axillary line 8-mm trocar site. Small bowel herniation through the same site as the first case was found in the second case. Emergency exploration was performed in both cases by extending the left trocar site. There was no sign of bowel ischemia, and successful bowel reduction and hernia repair were done. Unlike previously reported cases, these cases occurred in a normal body mass index (BMI) patient (first case 20.28 kg/m2, second case BMI 24.80 kg/m2) and were pelvic drain insertion sites. These sites were the weak points of the abdominal muscle coverage. Therefore, the closure of 8-mm trocar sites should be considered.

Fabrication and Characterization of Al2O3-Siloxane Composite Thermal Pads for Thermal Interface Materials.

In this study, Al2O3-siloxane composite thermal pads were fabricated using a tape-casting technique, and the thermal conductivity effect of the Al2O3 nanoparticle powder synthesized using a flame fusion process on siloxane composite thermal pads was investigated. Furthermore, various case studies were implemented, wherein the synthesized Al2O3 nanoparticle powder was subjected to different surface treatments, including dehydration, decarbonization, and silylation, to obtain Al2O3-siloxane composite thermal pads with high thermal conductivity. The experimental results confirmed that the thermal conductivity of the Al2O3-siloxane composite pads improved when fabricated using surface-treated Al2O3 nanoparticle powder synthesized with an optimally spheroidized crystal structure compared to that produced using non-treated Al2O3 nanoparticle powder. Therefore, this study provides guidelines for fabricating Al2O3-siloxane composite thermal pads with high thermal conductivity in the field of thermal interface materials.

Altered Sexual Response-Related Functional Connectivity and Morphometric Changes Influenced by Sex Hormones across Menopausal Status.

Our study retrospectively investigated differential patterns of the functional connectivity (FC) of core brain regions synchronous with morphometric changes associated with sexual dysfunction in menopausal women, and their correlations with sexual hormones. Twenty-three premenopausal women (mean age: 41.52 ± 7.38 years) and 21 menopausal women (mean age: 55.52 ± 2.80 years) underwent sex hormone level measurements with high-resolution T1 and functional magnetic resonance imaging (MRI) during rest, neutral, and sexual arousal conditions. Analysis of covariance adjusted for age was used to compare the FC and gray matter (GM) volume between the two groups. Menopausal women showed lower GM volumes in the superior frontal gyrus (SFG), superior temporal pole, parahippocampal gyrus (PHG), hippocampus (Hip), amygdala (Amg), and cerebellum (Cb) compared to premenopausal women (p < 0.05). In addition, compared to premenopausal women, menopausal women showed decreased FC of seed regions involved in the SFG, frontal eye fields, and Amg, as well as target regions involved in the PHG, Hip, inferior frontal gyrus, Cb, and vermis (p < 0.005). Furthermore, the FC between the right Amg and right Cb and between the left Amg and right Cb during sexual arousal in both groups was positively correlated with total estrogen and estradiol levels, respectively (p < 0.01). The GM volume values in the right Amg and right Cb were positively correlated with total estrogen and estradiol levels (p < 0.05). Our study demonstrated an association between menopause-related differential FC and GM volume variations and fluctuating sex hormones. Our findings highlight that overlapping brain regions with functional alterations and morphometric changes are closely linked with menopausal symptom-related decreases in sexual arousal and hormone levels.

A Successful Live Birth From a Vitrified Oocyte for Fertility Preservation of a Patient With Borderline Ovarian Tumor Undergoing Bilateral Ovarian Surgery: A Case Report.

This article reports the live birth of a healthy newborn using vitrified-warmed oocytes from fertility preservation before ovarian surgery. The patient in our case underwent two cycles of controlled ovarian stimulation before laparoscopic bilateral ovarian cystectomy for endometriosis, and a total of 23 mature oocytes were vitrified. After surgery, her pathologic reports revealed a serous borderline tumor and endometrioma. Fifteen months after her second surgery of laparoscopic right salpingo-oophorectomy and left ovarian cystectomy owing to recurrence, she had been married by then, and three of the frozen oocytes were thawed for intracytoplasmic sperm injection. These oocytes were cryopreserved for 2.5 years. All three were fertilized, and two grade-A cleavage-stage embryos were transferred. A singleton pregnancy was achieved, resulting in the delivery of a healthy baby boy at 39.3 weeks of gestation. Oocyte cryopreservation is an effective method for fertility preservation prior to ovarian surgery when ovarian function decline is predictable.

Prevalence and confounders of chronic endometritis diagnosed using CD138 in patients with recurrent implantation failure.

OBJECTIVE: This retrospective study aimed to investigate the prevalence of chronic endometritis, diagnosed using CD138 immunohistochemistry, among infertile women and to assess the association between chronic endometritis and recurrent implantation failure (RIF). METHODS: In total, 266 patients who underwent hysteroscopy due to infertility between 2019 and 2020 were included in the analysis. Of these, 136 patients with RIF and 130 non-RIF patients were included in the study. CD138 immunohistochemistry test results, blood biomarkers (including natural killer cells, white blood cells, and the lymphocyte-to-neutrophil ratio), and data on pregnancy outcomes were obtained. If the CD138 test yielded a positive result, the patients received antibiotic treatment. RESULTS: The overall proportion of CD138-positive patients was 32.7% (87/266). The CD138 positivity rate was not related to the number of cycles with implantation failure. In the RIF patient group, no significant associations were found between CD138 positivity and peripheral blood markers. The clinical pregnancy rates were similar between infertile women treated with antibiotics for chronic endometritis and those without chronic endometritis. CONCLUSION: To improve the pregnancy rate in infertile patients, it may be helpful to combine CD138 testing with other laboratory tests and administer antibiotic treatment if the result is positive.

SIRT1-dependent PGC-1α deacetylation by SRT1720 rescues progression of atherosclerosis by enhancing mitochondrial function.

Vascular smooth muscle cell (VSMC) senescence promotes atherosclerosis via lipid-mediated mitochondrial dysfunction and oxidative stress. However, the mechanisms of mitochondrial dysfunction and VSMC senescence in atherosclerosis have not been established. Here, we investigated the mechanisms whereby signaling pathways regulated by SRT1720 enhance or regulate mitochondrial functions in atherosclerotic VSMCs to suppress atherosclerosis. Initially, we examined the effect of SRT1720 on oleic acid (OA)-induced atherosclerosis. Atherosclerotic VSMCs exhibited elevated expressions of BODIPY and ADRP (adipose differentiation-related protein) and associated intracellular lipid droplet markers. In addition, the expression of collagen I was upregulated by OA, while the expressions of elastin and α-SMA were downregulated. mtDNA copy numbers, an ATP detection assay, transmission electron microscopy (TEM) imaging of mitochondria, mitochondria membrane potentials (assessed using JC-1 probe), and levels of mitochondrial oxidative phosphorylation (OXPHOS) were used to examine the effects of SRT1720 on OA-induced mitochondrial dysfunction. SRT1720 reduced mtDNA damage and accelerated mitochondria repair in VSMCs with OA-induced mitochondria dysfunction. In addition, mitochondrial reactive oxygen species (mtROS) levels were downregulated by SRT1720 in OA-treated VSMCs. Importantly, SRT1720 significantly increased SIRT1 and PGC-1α expression levels, but VSMCs senescence, inflammatory response, and atherosclerosis phenotypes were not recovered by treating cells with EX527 and SR-18292 before SRT1720. Mechanistically, the upregulations of SIRT1 and PGC-1α deacetylation by SRT1720 restored mitochondrial function, and consequently suppressed VSMC senescence and atherosclerosis-associated proteins and phenotypes. Collectively, this study indicates that SRT1720 can attenuate OA-induced atherosclerosis associated with VSMC senescence and mitochondrial dysfunction via SIRT1-mediated deacetylation of the PGC-1α pathway.

A Blend of Tamarindus Indica and Curcuma Longa Extracts Alleviates Monosodium Iodoacetate (MIA)-Induced Osteoarthritic Pain and Joint Inflammation in Rats.

BACKGROUND AND OBJECTIVE: NXT15906F6 (TamaFlexTM) is a proprietary herbal composition containing Tamarindus indica seeds and Curcuma longa rhizome extracts. NXT15906F6 supplementation has been shown clinically effective in reducing knee joint pain and improving musculoskeletal functions in healthy and knee osteoarthritis (OA) subjects. The objective of the present study was to assess the possible molecular basis of the anti-OA efficacy of NXT15906F6 in a monosodium iodoacetate (MIA)-induced model of OA in rats. METHODS: Healthy male Sprague Dawley rats (age: 8-9 wk body weight, B.W.: 225-308 g (n = 12) were randomly assigned to one of the six groups, (a) vehicle control, (b) MIA control, (c) Celecoxib (10 mg/kg B.W.), (d) TF-30 (30 mg/kg B.W.), (e) TF-60 (60 mg/kg B.W.), and (f) TF-100 (100 mg/kg B.W.). OA was induced by an intra-articular injection of 3 mg MIA into the right hind knee joint. The animals received either Celecoxib or TF through oral gavage over 28 days. The vehicle control animals received intra-articular sterile normal saline. RESULTS: Post-treatment, NXT15906F6 groups showed significant (p < 0.05) dose-dependent pain relief as evidenced by improved body weight-bearing capacity on the right hind limb. NXT15906F6 treatment also significantly reduced the serum tumor necrosis factor-α (TNF-α, p < 0.05) and nitrite (p < 0.05) levels in a dose-dependent manner. mRNA expression analyses revealed the up-regulation of collagen type-II (COL2A1) and down-regulation of matrix metalloproteinases (MMP-3, MMP-9 and MMP-13) in the cartilage tissues of NXT15906F6-supplemented rats. Cyclooxygenase-2 and inducible nitric oxide synthase (iNOS) protein expressions were down-regulated. Decreased immunolocalization of NF-κß (p65) was observed in the joint tissues of NXT15906F6-supplemented rats. Furthermore, microscopic observations revealed that NXT15906F6 preserved MIA-induced rats' joint architecture and integrity. CONCLUSION: NXT15906F6 reduces MIA-induced joint pain, inflammation, and cartilage degradation in rats.

Knockout of the lysosomal membrane protein, LAMP2C, improves transient gene expression in HEK293 cells via increased intracellular plasmid availability.

Plasmid-based transfection can be used in many applications such as transient gene expression (TGE)-based therapeutic protein production. These applications preferentially require maximization of intracellular plasmid availability. Here, we applied a lysosome engineering approach to alleviate lysosome-mediated nucleic acid degradation and enhance the TGE in mammalian cells. By knocking out the lysosomal membrane protein LAMP2C, which is known to be the main player in RNautophagy/DNautophagy (RDA), we significantly improved transient fluorescent protein expression in HEK293 cells by improving the retention rate of transfected plasmids; however, this effect was not observed in CHO cells. Additional knockout of a lysosomal membrane transporter and another RDA player, SIDT2, was ineffective, regardless of the presence of LAMP2C. LAMP2C knockout enhanced TGE-based mAb production in HEK293 cells by up to 2.82-fold increase in specific mAb productivity. Taken together, these results demonstrate that HEK293 cells can be engineered to improve the usage of the transfected plasmid via knockout of the lysosomal membrane protein LAMP2C and provide efficient host cells in TGE systems for therapeutic protein production.

Protective Effects of Repetitive Transcranial Magnetic Stimulation Against Streptozotocin-Induced Alzheimer's Disease.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation under investigation for treatment of a wide range of neurological disorders. In particular, the therapeutic application of rTMS for neurodegenerative diseases such as Alzheimer's disease (AD) is attracting attention. However, the mechanisms underlying the therapeutic efficacy of rTMS have not yet been elucidated, and few studies have systematically analyzed the stimulation parameters. In this study, we found that treatment with rTMS contributed to restoration of memory deficits by activating genes involved in synaptic plasticity and long-term memory. We evaluated changes in several intracellular signaling pathways in response to rTMS stimulation; rTMS treatment activated STAT, MAPK, Akt/p70S6K, and CREB signaling. We also systematically investigated the influence of rTMS parameters. We found an effective range of applications for rTMS and determined the optimal combination to achieve the highest efficiency. Moreover, application of rTMS inhibited the increase in cell death induced by hydrogen peroxide. These results suggest that rTMS treatment exerts a neuroprotective effect on cellular damage induced by oxidative stress, which plays an important role in the pathogenesis of neurological disorders. rTMS treatment attenuated streptozotocin (STZ)-mediated cell death and AD-like pathology in neuronal cells. In an animal model of sporadic AD caused by intracerebroventricular STZ injection, rTMS application improved cognitive decline and showed neuroprotective effects on hippocampal histology. Overall, this study will help in the design of stimulation protocols for rTMS application and presents a novel mechanism that may explain the therapeutic effects of rTMS in neurodegenerative diseases, including AD.

Diisobutyl phthalate (DiBP)-induced male germ cell toxicity and its alleviation approach.

Diisobutyl phthalate (DiBP) is a commonly used plasticizer in manufacturing consumer and industrial products to improve flexibility and durability. Despite of the numerous studies, however, the direct mechanism underlying the male reproductive damage of DiBP is poorly understood. In this study, we investigated the male germ cell toxicity of DiBP using GC-1 spermatogonia (spg) cells. Our results indicated that DiBP exposure causes oxidative stress and apoptosis in GC-1 spg cells. In addition, DiBP-derived autophagy activation and down-regulation of phosphoinositide 3-kinase (PI3K)-AKT and extracellular signal-regulated kinase (ERK) pathways further inhibited GC-1 spg cell proliferation, indicating that DiBP can instigate male germ cell toxicity by targeting several pathways. Importantly, a combined treatment of parthenolide, N-acetylcysteine, and 3-methyladenine significantly reduced DiBP-induced male germ cell toxicity and restored proliferation. Taken together, the results of this study can provide valuable information to the existing literature by enhancing the understanding of single phthalate DiBP-derived male germ cell toxicity and the therapeutic interventions that can mitigate DiBP damage.