Clinically relevant mutations in mycobacterial LepA cause rifampicin-specific phenotypic resistance.

Although all wild-type bacterial populations exhibit antibiotic tolerance, bacterial mutants with higher or lower tolerant subpopulation sizes have been described. We recently showed that in mycobacteria, phenotypically-resistant subpopulations can grow in bulk-lethal concentrations of rifampicin, a first-line anti-tuberculous antibiotic targeting RNA polymerase. Phenotypic resistance was partly mediated by paradoxical upregulation of RNA polymerase in response to rifampicin. However, naturally occurring mutations that increase tolerance via this mechanism had not been previously described. Here, we used transposon insertional mutagenesis and deep sequencing (Tnseq) to investigate rifampicin-specific phenotypic resistance using two different in vitro models of rifampicin tolerance in Mycobacterium smegmatis. We identify multiple genetic factors that mediate susceptibility to rifampicin. Disruption of one gene, lepA, a translation-associated elongation factor, increased rifampicin tolerance in all experimental conditions. Deletion of lepA increased the subpopulation size that is able to grow in bulk-lethal rifampicin concentrations via upregulation of basal rpoB expression. Moreover, homologous mutations in lepA that are found in clinical Mycobacterium tuberculosis (Mtb) isolates phenocopy lepA deletion to varying degrees. Our study identifies multiple genetic factors associated with rifampicin tolerance in mycobacteria, and may allow correlation of genetic diversity of clinical Mtb isolates with clinically important phenotypes such as treatment regimen duration.

[Expression and Clinical Sgnificance of IL-6, IL-6R and MCL-1 in Patients with Multiple Myeloma].

OBJECTIVE: To study the expression level and clinical significance of serum interleukin-6 (IL-6), its receptor (IL-6R) and myeloid cell leukemin-1(MCL-1) in patients with multiple myeloma（MM）. METHODS: Ninety-eight cases of MM treated in our hospital from January 2014 to January 2019 were selected, and the patients were divided into three groups according to their DS stage: stage I (27 cases), stage II (34 cases) and stage III (37 cases). The expression levels of IL-6, IL-6R and MCL-1 in patients at different DS stages were compared, and the prognostic-related factors were analyzed. RESULTS: The expression levels of IL-6 and MCL-1 in patients rised with DS stages, and the difference showed statistical significance (P<0.05), but the level of IL-6R in three groups showed no significant difference (P>0.05). The prognosis of patients with different levels of IL-6 and MCL-1 was compared and the results were as follows, the median survival time of 41 patients with IL-6≥80 pg/ml was 33.0 months, and that of 57 patients with IL-6 <80 pg/ml was 33.5 months, which showed no significant difference (P>0.05). The median survival time of 45 patients with MCL-1≥200 pg/ml was 30.5 months, and that of 53 patients with MCL-1 <200 pg/ml was 37.0 months, and their difference was statistically significant (P<0.05). Sex, age, ß2-MG and Hb not significantly correlated with prognosis of patients (P>0.05), however, DS stage, IL-6 and MCL-1 correlated with prognosis of patients（r=2.261，r=1.754，r=1.905）. CONCLUSION: The levels of IL-6 and MCL-1 in patients with multiple myeloma correlate with the DS stage and prognosis of patients.

Rifampicin can induce antibiotic tolerance in mycobacteria via paradoxical changes in rpoB transcription.

Metrics commonly used to describe antibiotic efficacy rely on measurements performed on bacterial populations. However, certain cells in a bacterial population can continue to grow and divide, even at antibiotic concentrations that kill the majority of cells, in a phenomenon known as antibiotic tolerance. Here, we describe a form of semi-heritable tolerance to the key anti-mycobacterial agent rifampicin, which is known to inhibit transcription by targeting the ß subunit of the RNA polymerase (RpoB). We show that rifampicin exposure results in rpoB upregulation in a sub-population of cells, followed by growth. More specifically, rifampicin preferentially inhibits one of the two rpoB promoters (promoter I), allowing increased rpoB expression from a second promoter (promoter II), and thus triggering growth. Disruption of promoter architecture leads to differences in rifampicin susceptibility of the population, confirming the contribution of rifampicin-induced rpoB expression to tolerance.

[Mycoplasma genitalium infection in patients attending the STD clinic in Nanjing].

OBJECTIVE: To analyze Mycoplasma genitalium (MG) infection among the patients attending the clinic of sexually transmitted diseases (STD) in Nanjing. METHODS: Urethral and cervical swabs were collected from 2 753 patients (2 161 males and 592 females) who first sought medical care at our STD Clinic from November 2015 to December 2017. The patients ranged in age from 18 to 67 years (ï¼»37.55 ± 10.37ï¼½ yr), divided into six age groups: ≤20, 21－30, 31－40, 41－50, 51－60, and >60 yr. The samples were examined for MG infection by simultaneous amplification and testing, Chlamydia trachomatis (CT) by quantitative real-time PCR, Neisseria gonorrhoeae (NG), Ureaplasma urealyticum (UU) and Mycoplasma hominis (MH) with the Thayer-Martin medium, and the count of polymorphonuclear leukocytes (PMNL) by microscopy with Methylene blue stain. RESULTS: Among the 2 753 samples, 219 (7.95%), including 176 males (8.14%) and 43 females (7.26%), were found positive for MG, with no statistically significant differences between the male and female groups (χ2 = 0.492, P = 0.483). The MG infection rates in the male and female groups were 6.67% vs 12.12% in the ≤20-year-olds, 8.44% vs 8.64% in the 21－30-year-olds, 7.63% vs 6.19% in the 31－40-year-olds, 10% vs 4.72% in the 41－50-year-olds, 5.64% vs 0 in the 51－60-year-olds, and 8.33% vs 0 in the >60-year-olds, with no statistically significant differences among the age groups (χ2 = 4.76, P = 0.446), or in the males (χ2 = 7.240, P = 0.200) or females (χ2 = 6.718, P = 0.076). The incidence rate of MG simple infection was markedly higher in the males than in the females (62.30% ï¼»76/122ï¼½ vs 36.84% ï¼»14/38ï¼½, χ2 = 7.041, P < 0.01). MG infection was found in combination with one or more pathogens like NG, CT, UU and MH, with MG+UU as the most common co-infection (21.31% ï¼»26/122ï¼½ in males and 31.85% ï¼»12/38ï¼½ in females). Of the 76 male patients with MG simple infection, 30 (39.47%) had ≥5 PMNLs per high-power field, and 66 (86.84%) showed symptoms of urethritis. CONCLUSIONS: MG infection was found in both the symptomatic and asymptomatic patients attending the STD clinic in Nanjing, with no significant difference in the incidence rate between males and females. A higher rate of MG simple infection was observed in the males than in the females, most of the male patients with symptoms of urethritis and urethral PMNLs.

Protective effect of rosiglitazone against acetaminophen-induced acute liver injury is associated with down-regulation of hepatic NADPH oxidases.

The peroxisome proliferator-activated receptor gamma (PPAR-γ) is a ligand-activated nuclear receptor that regulates glucose and lipid metabolism. The aim of the present study was to investigate the effects of rosiglitazone (RSG), a synthetic PPAR-γ agonist, on acetaminophen (APAP)-induced acute liver injury. Male CD-1 mice were injected with APAP (300mg/kg). Some mice were pretreated with RSG (20mg/kg) 48, 24 and 1h before APAP injection. As expected, RSG pretreatment alleviated APAP-induced acute liver injury. Moreover, RSG pretreatment attenuated APAP-induced hepatic cell death and improved the survival. Although it did not affect hepatic cytochrome P450 (CYP)2E1 expression, RSG pretreatment attenuated reduction of hepatic glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rd) and glutathione S-transferase (GST) activities, inhibited upregulation of hepatic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-2 and NOX-4, and alleviated hepatic GSH depletion during APAP-induced acute liver injury. In addition, RSG pretreatment suppressed activation of hepatic nuclear factor kappa B (NF-κB) and extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling during APAP-induced acute liver injury. These results provide a novel mechanistic explanation for RSG-mediated protection against APAP-induced acute liver injury. The present results suggest that synthetic PPAR-γ agonists might be effective agents for preventing the progression of APAP-induced acute liver injury.

Obeticholic acid protects against carbon tetrachloride-induced acute liver injury and inflammation.

The farnesoid X receptor (FXR) is a ligand-activated transcription factor that plays important roles in regulating bile acid homeostasis. The aim of the present study was to investigate the effects of obeticholic acid (OCA), a novel synthetic FXR agonist, carbon tetrachloride (CCl4)-induced acute liver injury. Mice were intraperitoneally injected with CCl4 (0.15ml/kg). In CCl4+OCA group, mice were orally with OCA (5mg/kg) 48, 24 and 1h before CCl4. As expected, hepatic FXR was activated by OCA. Interestingly, OCA pretreatment alleviated CCl4-induced elevation of serum ALT and hepatic necrosis. Moreover, OCA pretreatment inhibited CCl4-induced hepatocyte apoptosis. Additional experiment showed that OCA inhibits CCl4-induced hepatic chemokine gene Mcp-1, Mip-2 and Kc. Moreover, OCA inhibits CCl4-induced hepatic pro-inflammatory gene Tnf-α and Il-1ß. By contrast, OCA pretreatment elevated hepatic anti-inflammatory gene Il-4. Further analysis showed that OCA pretreatment inhibited hepatic IκB phosphorylation and blocked nuclear translocation of NF-κB p65 and p50 subunits during CCl4-induced acute liver injury. In addition, OCA pretreatment inhibited hepatic Akt, ERK and p38 phosphorylation in CCl4-induced acute liver injury. These results suggest that OCA protects against CCl4-induced acute liver injury and inflammation. Synthetic FXR agonists may be effective antidotes for hepatic inflammation during acute liver injury.

Vitamin d deficiency attenuates high-fat diet-induced hyperinsulinemia and hepatic lipid accumulation in male mice.

It is increasingly recognized that vitamin D deficiency is associated with increased risks of metabolic disorders among overweight children. A recent study showed that vitamin D deficiency exacerbated inflammation in nonalcoholic fatty liver disease through activating toll-like receptor 4 in a high-fat diet (HFD) rat model. The present study aimed to further investigate the effects of vitamin D deficiency on HFD-induced insulin resistance and hepatic lipid accumulation. Male ICR mice (35 d old) were randomly assigned into 4 groups as follows. In control diet and vitamin D deficiency diet (VDD) groups, mice were fed with purified diets. In HFD and VDD+HFD groups, mice were fed with HFD. In VDD and VDD+HFD groups, vitamin D in feed was depleted. Feeding mice with vitamin D deficiency diet did not induce obesity, insulin resistance, and hepatic lipid accumulation. By contrary, vitamin D deficiency markedly alleviated HFD-induced overweight, hyperinsulinemia, and hepatic lipid accumulation. Moreover, vitamin D deficiency significantly attenuated HFD-induced up-regulation of hepatic peroxisome proliferator-activated receptor γ, which promoted hepatic lipid uptake and lipid droplet formation, and its target gene cluster of differentiation 36. In addition, vitamin D deficiency up-regulated carnitine palmitoyltrans 2, the key enzyme for fatty acid ß-oxidation, and uncoupling protein 3, which separated oxidative phosphorylation from ATP production, in adipose tissue. These data suggest that vitamin D deficiency is not a direct risk factor for obesity, insulin resistance, and hepatic lipid accumulation. Vitamin D deficiency alleviates HFD-induced overweight, hyperinsulinemia, and hepatic lipid accumulation through promoting fatty acid ß-oxidation and elevating energy expenditure in adipose tissue.

Bio-relevant cobalt(II) complexes with compartmental polyquinoline ligand: synthesis, crystal structures and biological activities.

Three new Co(II) complexes, [Co4(L)2(µ3-CrO4)2](ClO4)2·2CH3CN (1), [Co2(L)(µ2-na)(H2O)](ClO4)2 (2) and [Co2(L)(µ2-ba)](ClO4)2·0.5CH3CN (3) (Hna=nicotinic acid, Hba=benzoic acid, HL=N,N,N',N'-tetrakis (2-quinolylmethyl)-1,3-diaminopropan-2-ol), have been synthesized and characterized by various physicochemical techniques. The Co(II) centers are connected by endogenous alkoxy bridge from L(-) and various extrinsic auxiliary linkers, some of which display coordination number asymmetry (5, 6-coordinated for 1 and 2; 5, 5-coordinated for 3). It is worth mentioning that complex 1 contains two rare reported µ3-η(1), η(1), η(1)-CrO4(2-) moieties. Susceptibility data of three complexes indicated intramolecular antiferromagnetic coupling of high-spin Co(II) atoms with exchange integral values (J) -14.94 cm(-1), -11.26 cm(-1) and -13.66 cm(-1) for 1, 2 and 3, respectively. Interaction of compounds with calf thymus DNA (CT-DNA) have been investigated by absorption spectral titration, ethidium bromide (EB) displacement assay and viscosity measurement, which revealed that compounds bound to CT-DNA with a moderate intercalative mode, accompanied the affinities order: 1>2≈3. Three complexes exhibit oxidative cleavage of pBR322 plasmid DNA including a reliance on H2O2 as the activator. Compound 1 demonstrates an increased DNA cleavage activity as compared with 2 and 3, which could degrade super coiled DNA (SC DNA) into nicked coiled DNA (NC DNA) in lower concentration (5 µM). Moreover, all compounds could quench the intrinsic fluorescence of bovine serum albumin (BSA) in a static quenching process. Complex 1 also shows higher anticancer activity than cisplatin with lower IC50 value of incubation for both 24 h and 48 h.

Synthesis, crystal structure, and biological activities of two chiral mononuclear Mn((III)) complexes.

Two new chiral mononuclear Mn((III)) complexes, [MnL((R)) Cl (C2 H5 OH)]•C2 H5 OH () and [MnL((S)) (CH3 OH)2 ]Cl•CH3 OH (), {H2 L = (R,R)-or (S,S)-N,N'-bis-(2-hydroxy-1-naphthalidehydene)-cyclohexanediamine} were synthesized and characterized by various physicochemical techniques. Bond valence sum (BVS) calculations and the Jahn-Teller effect indicate that the Mn centers are in a +3 oxidation state. The statuses of the two complexes in the solution were confirmed as a pair of enantiomers by electrospray ionization, mass spectrometry (ESI-MS) spectrum. The binding ability of the complexes with calf thymus CT-DNA was investigated by spectroscopic and viscosity measurements. Both of the complexes could interact with CT-DNA via an intercalative mode with the order of (R-enantiomer) > (S-enantiomer). Under the physiological conditions, the two compounds exhibit efficient DNA cleavage activities without any external agent, which also follows the order of R-enantiomer > S-enantiomer. Interestingly, the concentration-dependent DNA cleavage experiments indicate an optimal concentration of 17.5 µM. In addition, the interaction of the compounds with bovine serum albumin (BSA) was also investigated, which indicated that the complexes could quench the intrinsic fluorescence of BSA by a static quenching mechanism.

Effects of maternal LPS exposure during pregnancy on metabolic phenotypes in female offspring.

It is increasingly recognized that intra-uterine growth restriction (IUGR) is associated with an increased risk of metabolic disorders in late life. Previous studies showed that mice exposed to LPS in late gestation induced fetal IUGR. The present study investigated the effects of maternal LPS exposure during pregnancy on metabolic phenotypes in female adult offspring. Pregnant mice were intraperitoneally injected with LPS (50 µg/kg) daily from gestational day (GD)15 to GD17. After lactation, female pups were fed with standard-chow diets (SD) or high-fat diets (HFD). Glucose tolerance test (GTT) and insulin tolerance test (ITT) were assessed 8 and 12 weeks after diet intervention. Hepatic triglyceride content was examined 12 weeks after diet intervention. As expected, maternal LPS exposure during pregnancy resulted in fetal IUGR. Although there was an increasing trend on fat mass in female offspring whose dams were exposed to LPS during pregnancy, maternal LPS exposure during pregnancy did not elevate the levels of fasting blood glucose and serum insulin and hepatic triglyceride content in female adult offspring. Moreover, maternal LPS exposure during pregnancy did not alter insulin sensitivity in adipose tissue and liver in female adult offspring. Further analysis showed that maternal LPS exposure during pregnancy did not exacerbate HFD-induced glucose tolerance and insulin resistance in female adult offspring. In addition, maternal LPS exposure during pregnancy did not aggravate HFD-induced elevation of hepatic triglyceride content in female adult offspring. In conclusion, LPS-induced IUGR does not alter metabolic phenotypes in adulthood.

Maternal LPS exposure during pregnancy impairs testicular development, steroidogenesis and spermatogenesis in male offspring.

Lipopolysaccharide (LPS) is associated with adverse developmental outcomes including embryonic resorption, fetal death, congenital teratogenesis and fetal growth retardation. Here, we explored the effects of maternal LPS exposure during pregnancy on testicular development, steroidogenesis and spermatogenesis in male offspring. The pregnant mice were intraperitoneally injected with LPS (50 µg/kg) daily from gestational day (GD) 13 to GD 17. At fetal period, a significant decrease in body weight and abnormal Leydig cell aggregations were observed in males whose mothers were exposed to LPS during pregnancy. At postnatal day (PND) 26, anogenital distance (AGD), a sensitive index of altered androgen action, was markedly reduced in male pups whose mothers were exposed to LPS daily from GD13 to GD 17. At PND35, the weight of testes, prostates and seminal vesicles, and serum testosterone (T) level were significantly decreased in LPS-treated male pups. At adulthood, the number of sperm was significantly decreased in male offspring whose mothers were exposed to LPS on GD 13-17. Maternal LPS exposure during gestation obviously diminished the percent of seminiferous tubules in stages I-VI, increased the percent of seminiferous tubules in stages IX-XII, and caused massive sloughing of germ cells in seminiferous tubules in mouse testes. Moreover, maternal LPS exposure significantly reduced serum T level in male mice whose mothers were exposed to LPS challenge during pregnancy. Taken together, these results suggest that maternal LPS exposure during pregnancy disrupts T production. The decreased T synthesis might be associated with LPS-induced impairments for spermatogenesis in male offspring.