Epidemiology of Murine Typhus in Taiwan from 2013 to 2020.

Murine typhus is a flea-borne disease caused by Rickettsia typhi infection. The disease is a notifiable infectious disease in Taiwan. Specimens from suspected cases are required to be sent to the Taiwan Centers for Disease Control and Prevention for laboratory diagnosis. In this study, 204 cases of murine typhus were identified by bacterial isolation, real-time polymerase chain reaction, or indirect immunofluorescence assay between 2013 and 2020. The average incidence rate was 0.11/100,000 person-years (95% CI: 0.08-0.13). Murine typhus occurred throughout the year, but it was most prevalent in summer (May to August). The majority of patients were males (75%), residents of Kaohsiung city (31%), and worked in agriculture, forestry, fishing, and animal husbandry (27%). Fever was the most common symptom, present in 95.6% of patients, followed by headache (41%), myalgia (33%), and liver dysfunction (33%). Only 13% of patients had a rash. Up to 80% of cases were among hospitalized patients, and 43% of patients developed severe manifestations. Serological assays also indicated coinfection events. Seven patients showed a 4-fold increase in antibody titers against Orientia tsutsugamushi (N = 2), Coxiella burnetii (n = 2), and Leptospira (N = 3). In conclusion, murine typhus is an endemic and important zoonotic rickettsial disease in Taiwan that cannot be ignored. Further epidemiological surveillance and clinical characteristics should be continuously investigated to prevent and control murine typhus.

A ubiquitous endocrine disruptor tributyltin induces muscle wasting and retards muscle regeneration.

BACKGROUND: Organotin pollutant tributyltin (TBT) is an environmental endocrine disrupting chemical and is a known obesogen and diabetogen. TBT can be detected in human following consumption of contaminated seafood or water. The decrease in muscle strength and quality has been shown to be associated with type 2 diabetes in older adults. However, the adverse effects of TBT on the muscle mass and function still remain unclear. Here, we investigated the effects and molecule mechanisms of low-dose TBT on skeletal muscle regeneration and atrophy/wasting using the cultured skeletal muscle cell and adult mouse models. METHODS: The mouse myoblasts (C2C12) and differentiated myotubes were used to assess the in vitro effects of low-dose tributyltin (0.01-0.5 µM). The in vivo effects of TBT at the doses of 5 and 25 µg/kg/day (n = 6/group), which were five times lower than the established no observed adverse effect level (NOAEL) and equal to NOAEL, respectively, by oral administration for 4 weeks on muscle wasting and muscle regeneration were evaluated in a mouse model with or without glycerol-induced muscle injury/regeneration. RESULTS: TBT reduced myogenic differentiation in myoblasts (myotube with 6-10 nuclei: 53.9 and 35.8% control for 0.05 and 0.1 µM, respectively, n = 4, P < 0.05). TBT also decreased myotube diameter, upregulated protein expression levels of muscle-specific ubiquitin ligases (Atrogin-1 and MuRF1), myostatin, phosphorylated AMPKα, and phosphorylated NFκB-p65, and downregulated protein expression levels of phosphorylated AKT and phosphorylated FoxO1 in myotubes (0.2 and 0.5 µM, n = 6, P < 0.05). Exposure of TBT in mice elevated body weight, decreased muscle mass, and induced muscular dysfunction (5 and 25 µg/kg, P > 0.05 and P < 0.05, respectively, n = 6). TBT inhibited soleus muscle regeneration in mice with glycerol-induced muscle injury (5 and 25 µg/kg, P > 0.05 and P < 0.05, respectively, n = 6). TBT upregulated protein expression levels of Atrogin-1, MuRF1, myostatin, and phosphorylated AMPKα and downregulated protein expression level of phosphorylated FoxO1 in the mouse soleus muscles (5 and 25 µg/kg, P > 0.05 and P < 0.05, respectively, n = 6). CONCLUSIONS: This study demonstrates for the first time that low-dose TBT significantly inhibits myogenic differentiation and triggers myotube atrophy in a cell model and significantly decreases muscle regeneration and muscle mass and function in a mouse model. These findings suggest that low-dose TBT exposure may be an environmental risk factor for muscle regeneration inhibition, atrophy/wasting, and disease-related myopathy.

Benzo[a]pyrene alters vascular function in rat aortas ex vivo and in vivo.

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon found in tobacco smoke and air pollution products. BaP exposure has been recently suggested to be a risk factor for hypertension in coke oven workers. The mechanisms of BaP on vascular smooth muscle function remain unclear. Here, we examined the influence and possible mechanism of BaP on vasoconstriction in rat thoracic aortas ex vivo and in vivo. In vivo exposure of rats to BaP (20 mg/kg) for 8 weeks caused a significant enhancement in the systolic blood pressure and enhanced aortic hyperreactivity to α1-adrenoceptor selective agonist phenylephrine in aortas. BaP (1 and 10 µM) treatment for 18 h induced an enhancement of phenylephrine-induced vasoconstriction in the organ cultures of aortas. Aryl hydrocarbon receptor antagonist α-naphthoflavone, protein kinase C (PKC) inhibitor chelerythrine, mitogen-activated protein kinases (MAPK) inhibitor PD98059, myosin light chain kinase (MLCK) inhibitor ML-9, and Rho-kinase inhibitor Y-27632 significantly suppressed BaP-enhanced vasoconstriction. BaP time-dependently triggered reactive oxygen species (ROS) production in primary vascular smooth muscle cells. Both antioxidant N-acetylcysteine and NAD(P)H oxidase inhibitor diphenyleneiodonium significantly inhibited BaP-triggered ROS production and vasoconstriction. These results suggest that BaP enhances aortic vasoconstriction via the activation of ROS and muscular signaling molecules PKC, MAPK, MLCK, and Rho-kinase.

Acute oral toxicity and repeated dose 28-day oral toxicity studies of MIL-101 nanoparticles.

Metal-organic frameworks (MOFs) nanoparticles are a class of porous crystalline materials constructed from the bonding metal ions or clusters linked with organic ligands to form frameworks. MIL-101(Cr), one of the most representative MOFs, is a three-dimensional chromium terephthalate-base porous material consisted of chromium (III)-trimers cross-linked by 1,4-benzene dicarboxylate. The present study focused on determining the safety of MIL-101(Cr) nanoparticle. The acute oral toxicity and 28-day oral toxicity in mice were investigated. An acute oral toxicity test of MIL-101(Cr) nanoparticle for female mice showed that no mortality or any significant change observed at 2000 mg/kg body weight. A dose-dependent 28-day oral toxicity evaluation of MIL-101(Cr) nanoparticle for male and female mice revealed no significant effects on mortality, feed consumption, body weight, organ weight, and behavior. Assessments of hematology, clinical biochemistry, and histopathology revealed no adverse effects in mice treated with MIL-101(Cr) nanoparticle (10-1000 mg/kg). These results suggest that MIL-101(Cr) nanoparticle has no significant acute and subacute toxicity. The no observed adverse effect level of MIL-101(Cr) nanoparticle was defined as at least 1000 mg/kg/day orally for 28 days for male and female mice.

Prenatal developmental toxicity study of strontium citrate in Sprague Dawley rats.

In this study, the pregnant female Sprague Dawley (SD) rats were used to evaluate the potential toxicological effect of strontium citrate, a dietary supplement, on embryo-fetal development. Strontium citrate at doses of 0 mg/kg, 680 mg/kg, 1360 mg/kg, and 2267 mg/kg was administrated orally by gavage to rats at day 6 to day 15 of pregnancy. Each group contained 20 pregnant rats. On the 20th day of gestation, rats was anesthetized and dissected by cesarean section. The appearance, internal organs, gravid uterus weight, embryo implantation number, and implantation loss rate in maternal rats of each group did not reveal any lesions. In fetuses, there were no statistical differences in the fetus weight, sex ratio, embryo resorption number, stillbirth number, and fetal visceral examination in all testing groups compared to the control group. However, in 2267 mg/kg strontium citrate group, the fetuses showed the statistical differences in the anomalies of the bones and eyes compared to the control group. These findings indicate that high-dose strontium citrate possesses an adverse effect on embryonic and fetal development in SD rats. The no observed adverse effect level (NOAEL) of strontium citrate for prenatal development toxicity in SD rats may be regarded as 1360 mg/kg/day.

Preventing muscle wasting by osteoporosis drug alendronate in vitro and in myopathy models via sirtuin-3 down-regulation.

BACKGROUND: A global consensus on the loss of skeletal muscle mass and function in humans refers as sarcopenia and cachexia including diabetes, obesity, renal failure, and osteoporosis. Despite a current improvement of sarcopenia or cachexia with exercise training and supportive therapies, alternative and specific managements are needed to discover for whom are unable or unwilling to embark on these treatments. Alendronate is a widely used drug for osteoporosis in the elderly and postmenopausal women. Osteopenic menopausal women with 6 months of alendronate therapy have been observed to improve not only lumbar bone mineral density but also handgrip strength. However, the effect and mechanism of alendronate on muscle strength still remain unclear. Here, we investigated the therapeutic potential and the molecular mechanism of alendronate on the loss of muscle mass and strength in vitro and in vivo. METHODS: Mouse myoblasts and primary human skeletal muscle-derived progenitor cells were used to assess the effects of low-dose alendronate (0.1-1 µM) combined with or without dexamethasone on myotube hypertrophy and myogenic differentiation. Moreover, we also evaluated the effects of low-dose alendronate (0.5 and 1 mg/kg) by oral administration on the limb muscle function and morphology of mice with denervation-induced muscle atrophy and glycerol-induced muscle injury. RESULTS: Alendronate inhibited dexamethasone-induced myotube atrophy and myogenic differentiation inhibition in mouse myoblasts and primary human skeletal muscle-derived progenitor cells. Alendronate significantly abrogated dexamethasone-up-regulated sirtuin-3 (SIRT3), but not SIRT1, protein expression in myotubes. Both SIRT3 inhibitor AKG7 and SIRT3-siRNA transfection could also reverse dexamethasone-up-regulated atrogin-1 and SIRT3 protein expressions. Animal studies showed that low-dose alendronate by oral administration ameliorated the muscular malfunction in mouse models of denervation-induced muscle atrophy and glycerol-induced muscle injury with a negative regulation of SIRT3 expression. CONCLUSIONS: The putative mechanism by which muscle atrophy was improved with alendronate might be through the SIRT3 down-regulation. These findings suggest that alendronate can be a promising therapeutic strategy for management of muscle wasting-related diseases and sarcopenia.

Down-regulation of MSH2 expression by Hsp90 inhibition enhances cytotoxicity affected by tamoxifen in human lung cancer cells.

The anti-estrogen tamoxifen has been used worldwide as an adjuvant hormone therapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Human MutS homolog 2 (MSH2), a crucial element of the highly conserved DNA mismatch repair system, and expression of MSH2 have been down-regulated by Hsp90 function inhibition in human lung cancer. Therefore, in this study, we examined whether MSH2 plays a role in the tamoxifen and Hsp90 inhibitor-induced cytotoxic effect on NSCLC cells. The results showed that treatment with tamoxifen increased MSH2 mRNA and protein levels. The combination treatment with PI3K inhibitors (LY294002 or wortmannin) or knockdown AKT expression by specific small interfering RNA could decrease tamoxifen-induced MSH2 expression. Both knocking down MSH2 expression and co-treatment of PI3K inhibitors enhanced the cytotoxicity and cell growth inhibition of tamoxifen. Compared to a single agent alone, tamoxifen combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced MSH2 expression. These findings may have implications for the rational design of future drug regimens incorporating tamoxifen and Hsp90 inhibitors for the treatment of NSCLC.

Down-regulation of MSH2 expression by an Hsp90 inhibitor enhances pemetrexed-induced cytotoxicity in human non-small-cell lung cancer cells.

Elevated heat shock protein 90 (Hsp90) expression has been linked to poor prognosis in patients with non-small cell lung cancer (NSCLC). The multitargeted antifolate pemetrexed has demonstrated certain clinical activities against NSCLC. However, the efficacy of the combination of pemtrexed and Hsp90 inhibitor to prolong the survival of patients with NSCLC still remains unclear. Human MutS homolog 2 (MSH2), a crucial element of the highly conserved DNA mismatch repair system, and defects or polymorphisms of MSH2 have been found in lung cancer. In this study, we evaluated the effects of pemetrexed on NSCLC cell lines (H520 and H1703) and found that treatment with this drug at 20-50 µM increased the MSH2 mRNA and protein levels in a MKK3/6-p38 MAPK signal activation-dependent manner. Furthermore, the knockdown of MSH2 expression by transfection with small interfering RNA of MSH2 or the blockage of p38 MAPK activation by SB202190 enhanced the cytotoxicity of pemetrexed. Combining the drug treatment with an Hsp90 inhibitor resulted in an enhanced pemetrexed-induced cytotoxic effect, accompanied with the reduction of MSH2 protein and mRNA levels. The expression of constitutively active MKK6 (MKK6E) or HA-p38 MAPK vectors significantly rescued the decreased p38 MAPK activity, and restored the MSH2 protein levels and cell survival in NSCLC cells co-treated with pemetrexed and Hsp90 inhibitor. In this study, we have demonstrated that down-regulation of the MKK3/6-p38 MAPK signal with the subsequent reduction of MSH2 enhanced the cytotoxic effect of pemetrexed in H520 and H1703 cells. The results suggest a potential future benefit of combining pemetrexed and the Hsp90 inhibitor to treat lung cancer.

Tamoxifen enhances erlotinib-induced cytotoxicity through down-regulating AKT-mediated thymidine phosphorylase expression in human non-small-cell lung cancer cells.

Tamoxifen is a triphenylethylene nonsteroidal estrogen receptor (ER) antagonist used worldwide as an adjuvant hormone therapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Thymidine phosphorylase (TP) is an enzyme of the pyrimidine salvage pathway which is upregulated in cancers. In this study, tamoxifen treatment inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with tamoxifen decreased TP mRNA and protein levels through AKT inactivation. Furthermore, expression of constitutively active AKT (AKT-CA) vectors significantly rescued the decreased TP protein and mRNA levels in tamoxifen-treated NSCLC cells. In contrast, combination treatment with PI3K inhibitors (LY294002 or wortmannin) and tamoxifen further decreased the TP expression and cell viability of NSCLC cells. Knocking down TP expression by transfection with small interfering RNA of TP enhanced the cytotoxicity and cell growth inhibition of tamoxifen. Erlotinib (Tarceva, OSI-774), an orally available small molecular inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for clinical treatment of NSCLC. Compared to a single agent alone, tamoxifen combined with erlotinib resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-AKT and phospho-ERK1/2, and reduced TP protein levels. These findings may have implications for the rational design of future drug regimens incorporating tamoxifen and erlotinib for the treatment of NSCLC.

Inhibition of p38 MAPK-dependent MutS homologue-2 (MSH2) expression by metformin enhances gefitinib-induced cytotoxicity in human squamous lung cancer cells.

OBJECTIVES: Gefitinib, a quinazoline-derived tyrosine kinase inhibitor, has anti-tumor activity in vivo and in vitro. Human MutS homologue-2 (MSH2) plays a central role in promoting genetic stability by correcting DNA replication errors. The present study investigated the effects of p38 mitogen-activated protein kinase (MAPK) signal on gefitinib-induced MSH2 expression in two human non-small cell lung squamous cancer cell lines. MATERIALS AND METHODS: After the gefitinib treatment, the expressions of MSH2 mRNA were determined by real-time PCR and RT-PCR analysis. Protein levels of MSH2, phospho-MKK3/6, phospho-p38 MAPK were determined by Western blot analysis. We used specific MSH2, and p38 MAPK small interfering RNA to examine the role of p38 MAPK-MSH2 signal in regulating the chemosensitivity of gefitinib. Cell viability was assessed by MTS assay, trypan blue exclusion, and colony-forming ability assay. RESULTS: Exposure of gefitinib increased MSH2 protein and mRNA levels, which was accompanied by MKK3/6-p38 MAPK activation in H520 and H1703 cells. Moreover, blocking p38 MAPK activation by SB202190 significantly decreased gefitinib-induced MSH2 expression by increasing mRNA and protein instability. In contrast, enhancing p38 activation using constitutively active MKK6 (MKK6E) increased MSH2 protein and mRNA levels. Specific inhibition of MSH2 expression by siRNA enhanced gefitinib-induced cytotoxicity. Metformin, an anti-diabetic drug, might reduce cancer risk. In human lung squamous cancer cells, metformin decreased gefitinib-induced MSH2 expression and augmented the cytotoxic effect and growth inhibition by gefitinib. Transient expression of MKK6E or HA-p38 MAPK vector could abrogate metformin and gefitinib-induced synergistic cytotoxic effect in H520 and H1703 cells. CONCLUSION: Together, down-regulation of MSH2 expression can be a possible strategy to enhance the sensitivity of gefitinib to human lung squamous cancer cells.

Pemetrexed downregulates ERCC1 expression and enhances cytotoxicity effected by resveratrol in human nonsmall cell lung cancer cells.

The multitargeted antifolate pemetrexed has demonstrated certain clinical activities against nonsmall cell lung cancer (NSCLC). Resveratrol (3,5,4-trihydroxy-trans-stilbene) is a polyphenol found in grapes and other plants and has great potential as a preventative and therapeutic agent due to its anticarcinogenic activity. The efficacy of adding resveratrol to pemetrexed to prolong the survival of patients with NSCLC still remains unclear. The excision repair cross-complementation 1 (ERCC1) is a DNA repair gene coding 5' endonuclease in nucleotide excision repair and is overexpressed in chemo- or radioresistant carcinomas. In this study, resveratrol (10-50 µM) inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with resveratrol increased ERCC1 messenger RNA and protein levels in a MKK3/6-p38 MAPK signal activation-dependent manner. Furthermore, blocking p38 MAPK activation by SB202190 or knocking down ERCC1 expression by transfection with small interfering RNA of ERCC1 enhanced the cytotoxicity of resveratrol. Combining resveratrol with pemetrexed resulted in a synergistic cytotoxic effect, accompanied with the reduction of phospho-p38 MAPK and ERCC1 protein levels, and a DNA repair capacity. Expression of constitutively active MKK6 (MKK6E) or HA-p38 MAPK vectors significantly rescued the decreased p38 MAPK activity, and restored ERCC1 protein levels and cell survival in resveratrol and pemetrexed cotreated NSCLC cells. In this study, for the first time, we have demonstrated the synergistic effect of combined treatment with resveratrol and pemetrexed in human NSCLC cells through downregulation of the MKK3/6-p38 MAPK-ERCC1 signal, suggesting a potential benefit of combining resveratrol and pemetrexed to treat lung cancer in the future.

Metformin induces cytotoxicity by down-regulating thymidine phosphorylase and excision repair cross-complementation 1 expression in non-small cell lung cancer cells.

Metformin is an antidiabetic drug recently shown to inhibit cancer cell proliferation and growth, although the involved molecular mechanisms have not been elucidated. In many cancer cells, high expression of thymidine phosphorylase (TP) and Excision repair cross-complementation 1 (ERCC1) is associated with poor prognosis. We used A549 and H1975 human non-small cell lung cancer (NSCLC) cell lines to investigate the role of TP and ERCC1 expression in metformin-induced cytotoxicity. Metformin treatment decreased cellular TP and ERCC1 protein and mRNA levels by down-regulating phosphorylated MEK1/2-ERK1/2 protein levels in a dose- and time-dependent manner. The enforced expression of the constitutively active MEK1 (MEK1-CA) vectors significantly restored cellular TP and ERCC1 protein levels and cell viability. Specific inhibition of TP and ERCC1 expression by siRNA enhanced the metformin-induced cytotoxicity and growth inhibition. Arachidin-1, an antioxidant stilbenoid, further decreased TP and ERCC1 expression and augmented metformin's cytotoxic effect, which was abrogated in lung cancer cells transfected with MEK1/2-CA expression vector. In conclusion, metformin induces cytotoxicity by down-regulating TP and ERCC1 expression in NSCLC cells.

Metformin-mediated downregulation of p38 mitogen-activated protein kinase-dependent excision repair cross-complementing 1 decreases DNA repair capacity and sensitizes human lung cancer cells to paclitaxel.

Metformin, an extensively used and well-tolerated drug for treating individuals with type 2 diabetes, has recently gained significant attention as an anticancer drug. On the other hand, paclitaxel (Taxol) is a new antineoplastic drug that has shown promise in the treatment of non-small cell lung cancer (NSCLC). High expression levels of excision repair cross-complementary 1 (ERCC1) in cancers have been positively associated with the DNA repair capacity and a poor prognosis in NSCLC patients treated with platinum-containing chemotherapy. In this current study, paclitaxel was found to increase phosphorylation of mitogen-activated protein kinase (MAPK) kinase 3/6 (MKK3/6)-p38 MAPK as well as protein and mRNA levels of ERCC1 in H1650 and H1703 cells. Moreover, paclitaxel-induced ERCC1 protein and mRNA levels significantly decreased via the downregulation of p38 activity by either a p38 MAPK inhibitor SB202190 or p38 knockdown with specific small interfering RNA (siRNA). Specific inhibition of ERCC1 with siRNA was found to enhance the paclitaxel-induced cytotoxic effect and growth inhibition. Furthermore, metformin was able to not only decrease the paclitaxel-induced p38 MAPK-mediated ERCC1 expression, but also augment the cytotoxic effect induced by paclitaxel. Finally, expression of constitutive activate MKK6 or HA-p38 MAPK vectors in lung cancer cells was able to abrogate ERCC1 downregulation by metformin and paclitaxel as well as cell viability and DNA repair capacity. Overall, our results suggest that inhibition of the p38 MAPK signaling by metformin coupled with paclitaxel therapy in human NSCLC cells may be a clinically useful combination, which however will require further validation.

HSP90 inhibition induces cytotoxicity via down-regulation of Rad51 expression and DNA repair capacity in non-small cell lung cancer cells.

Heat shock protein 90 (HSP90) is an exciting new target in cancer therapy. Repair protein Rad51 is involved in protecting non-small cell lung cancer (NSCLC) cell lines against chemotherapeutic agent-induced cytotoxicity. This study investigated the role of Rad51 expression in HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced cytotoxicity in two NSCLC cell lines, A549 and H1975. The 17-AAG treatment decreased cellular Rad51 protein and mRNA levels and phosphorylated MKK1/2-ERK1/2 protein levels, and disrupted the HSP90 and Rad51 interaction. This triggered Rad51 protein degradation through the 26S proteasome pathway. The 17-AAG treatment also decreased the NSCLC cells' DNA repair capacity, which was restored by the forced expression of the Flag-Rad51 vector. Specific inhibition of Rad51 expression by siRNA further enhanced 17-AAG-induced cytotoxicity. In contrast, enhanced ERK1/2 activation by the constitutively active MKK1/2 (MKK1/2-CA) vector significantly restored the 17-AAG-reduced Rad51 protein levels and cell viability. Arachidin-1, an antioxidant stilbenoid, further decreased Rad51 expression and augmented the cytotoxic effect and growth inhibition of 17-AAG. The 17-AAG and arachidin-1-induced synergistic cytotoxic effects and decreased DNA repair capacity were abrogated in lung cancer cells with MKK1/2-CA or Flag-Rad51 expression vector transfection. In conclusion, HSP90 inhibition induces cytotoxicity by down-regulating Rad51 expression and DNA repair capacity in NSCLC cells.