Intestinal epithelial dopamine receptor signaling drives sex-specific disease exacerbation in a mouse model of multiple sclerosis.

Although the gut microbiota can influence central nervous system (CNS) autoimmune diseases, the contribution of the intestinal epithelium to CNS autoimmunity is less clear. Here, we showed that intestinal epithelial dopamine D2 receptors (IEC DRD2) promoted sex-specific disease progression in an animal model of multiple sclerosis. Female mice lacking Drd2 selectively in intestinal epithelial cells showed a blunted inflammatory response in the CNS and reduced disease progression. In contrast, overexpression or activation of IEC DRD2 by phenylethylamine administration exacerbated disease severity. This was accompanied by altered lysozyme expression and gut microbiota composition, including reduced abundance of Lactobacillus species. Furthermore, treatment with N2-acetyl-L-lysine, a metabolite derived from Lactobacillus, suppressed microglial activation and neurodegeneration. Taken together, our study indicates that IEC DRD2 hyperactivity impacts gut microbial abundances and increases susceptibility to CNS autoimmune diseases in a female-biased manner, opening up future avenues for sex-specific interventions of CNS autoimmune diseases.

Differential Protein Expression in Set5p-Mediated Acetic Acid Stress Response and Novel Targets for Engineering Yeast Stress Tolerance.

Acetic acid is a prevalent inhibitor in lignocellulosic hydrolysate, which represses microbial growth and bioproduction. Histone modification and chromatin remodeling have been revealed to be critical for regulating eukaryotic metabolism. However, related studies in chronic acetic acid stress responses remain unclear. Our previous studies revealed that overexpression of the histone H4 methyltransferase Set5p enhanced acetic acid stress tolerance of the budding yeast Saccharomyces cerevisiae. In this study, we examined the role of Set5p in acetic acid stress by analyzing global protein expression. Significant activation of intracellular protein expression under the stress was discovered, and the functions of the differential proteins were mainly involved in chromatin modification, signal transduction, and carbohydrate metabolism. Notably, a substantial increase of Set5p expression was observed in response to acetic acid stress. Functional studies demonstrated that the restriction of the telomere capping protein Rtc3p, as well as Ies3p and Taf14p, which are related to chromatin regulation, was critical for yeast stress response. This study enriches the understanding of the epigenetic regulatory mechanisms underlying yeast stress response mediated by histone-modifying enzymes. The results also benefit the development of robust yeast strains for lignocellulosic bioconversion.

A systematic review and meta-analysis of the efficacy of ketamine and esketamine on suicidal ideation in treatment-resistant depression.

PURPOSE: To systematically assess the evidence of efficacy and safety of the use of ketamine and esketamine for patients with treatment-resistant depression (TRD) with suicidal ideation (SI). METHODS: We independently searched for clinical trials from inception to January 2023 using electronic databases, e.g., PubMed and EMBASE. A systematic review and meta-analysis were performed to assess SI scores of depression rating scales, which were regarded as the outcomes. RESULTS: A total of five independent double-blind, placebo controlled randomized clinical trials (RCTs) are eligible for inclusion. Four of the studies used ketamine as an intervention and one used esketamine as an intervention. Three hundred ninety-one patients with TRD were included (the intervention group with ketamine or esketamine is 246, and the control group is 145). No statistically significant interaction between the subscales of suicide ideation (SMD = - 0.66, 95% CI (- 1.61, 0.29); Z = 1.36, P = 0.17) and antidepressant effects (SMD = - 0.99, 95% CI (- 2.33, 0.34); Z = 1.46, P = 0.15) based on the results of ketamine and esketamine, compared with placebo groups. CONCLUSION: This meta-analysis suggested that esketamine and ketamine have failed to reduce suicidal ideation in patients with TRD. Further studies are desirable to confirm the effects of ketamine and esketamine in TRD patients.

UFMylation inhibits the proinflammatory capacity of interferon-γ-activated macrophages.

Macrophages activated with interferon-γ (IFN-γ) in combination with other proinflammatory stimuli, such as lipopolysaccharide or tumor necrosis factor-α (TNF-α), respond with transcriptional and cellular changes that enhance clearance of intracellular pathogens at the risk of damaging tissues. IFN-γ effects must therefore be carefully balanced with inhibitory mechanisms to prevent immunopathology. We performed a genome-wide CRISPR knockout screen in a macrophage cell line to identify negative regulators of IFN-γ responses. We discovered an unexpected role of the ubiquitin-fold modifier (Ufm1) conjugation system (herein UFMylation) in inhibiting responses to IFN-γ and lipopolysaccharide. Enhanced IFN-γ activation in UFMylation-deficient cells resulted in increased transcriptional responses to IFN-γ in a manner dependent on endoplasmic reticulum stress responses involving Ern1 and Xbp1. Furthermore, UFMylation in myeloid cells is required for resistance to influenza infection in mice, indicating that this pathway modulates in vivo responses to infection. These findings provide a genetic roadmap for the regulation of responses to a key mediator of cellular immunity and identify a molecular link between the UFMylation pathway and immune responses.

Astroglial Connexin 43-Mediated Gap Junctions and Hemichannels: Potential Antidepressant Mechanisms and the Link to Neuroinflammation.

Major depression disorder (MDD) is a neuropsychiatric disorder associated with a high suicide rate and a higher disability rate than any other disease. Evidence suggests that the pathological mechanism of MDD is related to astrocyte dysfunction. Depression is mainly associated with the expression of connexin 43 (Cx43) and the function of Cx43-mediated gap junctions and hemichannels in astrocytes. Moreover, neuroinflammation has been a hotspot in research on the pathology of depression, and Cx43-mediated functions are thought to be involved in neuroinflammation-related depression. However, the specific mechanism of Cx43-mediated functions in neuroinflammation-related depression pathology remains unclear. Therefore, this review summarizes and discusses Cx43 expression, the role of gap junction intercellular communication, and its relationship with neuroinflammation in depression. This review also focuses on the effects of antidepressant drugs (e.g., monoamine antidepressants, psychotropic drugs, and N-methyl-D-aspartate receptor antagonists) on Cx43-mediated function and provides evidence for Cx43 as a novel target for the treatment of MDD. The pathogenesis of MDD is related to astrocyte dysfunction, with reduced Cx43 expression, GJ dysfunction, decreased GJIC and reduced BDNF expression in the depressed brain. The effect of Cx43 on neuroinflammation-related depression involving inflammatory cytokines, glutamate excitotoxicity, and HPA axis dysregulation. Antidepressant drugs targeting Cx43 can effectively relieve depressive symptoms.

Glutamatergic receptor and neuroplasticity in depression: Implications for ketamine and rapastinel as the rapid-acting antidepressants.

PURPOSE OF REVIEW: To explore the convergent downstream pathways of ketamine and rapastinel and drive further development of identification for following generational rapid-acting antidepressants in the synaptic process. RECENT FINDINGS: Ketamine is an NMDAR antagonist and is proven effective in depression for the rapid and sustained antidepressant response, while rapastinel is an NMDAR positive allosteric modulator, producing antidepressant effects like ketamine with no severe side effects. The common antidepressant effects of ketamine and rapastinel are BDNF and mTORC1 pathway in synaptic plasticity.

CLEC3B is a novel causative gene for macular-retinal dystrophy.

PURPOSE: Macular degeneration is the leading cause of blindness worldwide. In this study, we aimed to define a new subtype of macular-retinal dystrophy and its genetic predisposition in 5 families. METHODS: Exome sequencing was performed to determine the putative disease-causing genes in patients with inherited macular disorders confirmed through comprehensive ophthalmic examinations. To validate its functional consequence, adeno-associated virus-mediated mutant gene was delivered into the murine retina, and both structural and functional tests were performed to investigate its pathological effects in vivo. RESULTS: In total, 5 multigenerational families diagnosed with autosomal dominant maculoretinopathy were found to carry a pathogenic variant in a new gene, CLEC3B, which encodes tetranectin, a plasminogen kringle-4 binding protein. Consistent with the disease phenotypes of patients, mice that received subretinal injections with the CLEC3B variant displayed multiple subretinal hyperreflective deposits, reduced retinal thickness, and decreased electroretinographic responses. Moreover, the optokinetic tracking response indicated that spatial frequency was significantly lower (P < .05), implying impaired visual function in these mice. CONCLUSION: We have presented a new subtype of macular-retinal dystrophy in 5 families as well as a new pathogenic gene, CLEC3B, providing new insights into maculoretinopathy etiology.

Mitophagy, a Form of Selective Autophagy, Plays an Essential Role in Mitochondrial Dynamics of Parkinson's Disease.

Parkinson's disease (PD) is a severe neurodegenerative disorder caused by the progressive loss of dopaminergic neurons in the substantia nigra and affects millions of people. Currently, mitochondrial dysfunction is considered as a central role in the pathogenesis of both sporadic and familial forms of PD. Mitophagy, a process that selectively targets damaged or redundant mitochondria to the lysosome for elimination via the autophagy devices, is crucial in preserving mitochondrial health. So far, aberrant mitophagy has been observed in the postmortem of PD patients and genetic or toxin-induced models of PD. Except for mitochondrial dysfunction, mitophagy is involved in regulating several other PD-related pathological mechanisms as well, e.g., oxidative stress and calcium imbalance. So far, the mitophagy mechanisms induced by PD-related proteins, PINK1 and Parkin, have been studied widely, and several other PD-associated genes, e.g., DJ-1, LRRK2, and alpha-synuclein, have been discovered to participate in the regulation of mitophagy as well, which further strengthens the link between mitophagy and PD. Thus, in this view, we reviewed mitophagy pathways in belief and discussed the interactions between mitophagy and several PD's pathological mechanisms and how PD-related genes modulate the mitophagy process.

Autophagy genes in myeloid cells counteract IFNγ-induced TNF-mediated cell death and fatal TNF-induced shock.

Host inflammatory responses must be tightly regulated to ensure effective immunity while limiting tissue injury. IFN gamma (IFNγ) primes macrophages to mount robust inflammatory responses. However, IFNγ also induces cell death, and the pathways that regulate IFNγ-induced cell death are incompletely understood. Using genome-wide CRISPR/Cas9 screening, we identified autophagy genes as central mediators of myeloid cell survival during the IFNγ response. Hypersensitivity of autophagy gene-deficient cells to IFNγ was mediated by tumor necrosis factor (TNF) signaling via receptor interacting protein kinase 1 (RIPK1)- and caspase 8-mediated cell death. Mice with myeloid cell-specific autophagy gene deficiency exhibited marked hypersensitivity to fatal systemic TNF administration. This increased mortality in myeloid autophagy gene-deficient mice required the IFNγ receptor, and mortality was completely reversed by pharmacologic inhibition of RIPK1 kinase activity. These findings provide insight into the mechanism of IFNγ-induced cell death via TNF, demonstrate a critical function of autophagy genes in promoting cell viability in the presence of inflammatory cytokines, and implicate this cell survival function in protection against mortality during the systemic inflammatory response.

A Cathode Interface Layer Based on 4,5,9,10-Pyrene Diimide for Highly Efficient Binary Organic Solar Cells.

Efficient cathode interfacial layers (CILs) are becoming essential elements for organic solar cells (OSCs). However, the absorption of commonly used cathode interfacial materials (CIMs) is either too weak or overlaps too much with that of photoactive materials, hindering their contribution to the light absorption. In this work, we demonstrate the construction of highly efficient CIMs based on 2,7-di-tert-butyl-4,5,9,10-pyrene diimide (t-PyDI) framework. By introducing amino, amino N-oxide and quaternary ammonium bromide as functional groups, three novel self-doped CIMs named t-PyDIN, t-PyDINO and t-PyDINBr are synthesized. These CIMs are capable of boosting the device performances by broadening the absorption, forming ohmic contact at the interface of active layer and electrode, as well as facilitating electron collection. Notably, the device based on t-PyDIN achieved a power conversion efficiency of 18.25 %, which is among the top efficiencies reported to date in binary OSCs.

Novel rapid-acting glutamatergic modulators: Targeting the synaptic plasticity in depression.

Major depressive disorder (MDD) is severely prevalent, and conventional monoaminergic antidepressants gradually exhibit low therapeutic efficiency, especially for patients with treatment-resistant depression. A neuroplasticity hypothesis is an emerging advancement in the mechanism of depression, mainly expressed in the glutamate system, e.g., glutamate receptors and signaling. Dysfunctional glutamatergic neurotransmission is currently considered to be closely associated with the pathophysiology of MDD. Biological function, pharmacological action, and signal attributes in the glutamate system both regulate the neural process. Specific functional subunits could be therapeutic targets to explore the novel glutamatergic modulators, which have fast-acting, and relatively sustained antidepressant effects. Here, the present review summarizes the pathophysiology of MDD found in the glutamate system, exploring the role of glutamate receptors and their downstream effects. These convergent mechanisms have prompted the development of other modulators targeting on glutamate system, including N-methyl-d-aspartate receptor antagonists, selective GluN2B-specific antagonists, glycine binding site agents, and regulators of metabotropic glutamate receptors. Relevant researches underly the putative mechanisms of these drugs, which reverse the damage of depression by regulating glutamatergic neurotransmission. It also provides further insight into the mechanism of depression and exploring potential targets for novel agent development.

Progress of tubulin polymerization activity detection methods.

Tubulin, an important target in tumor therapy, is one of the hotspots in the field of antineoplastic drugs in recent years, and it is of great significance to design and screen new inhibitors for this target. Natural products and chemical synthetic drugs are the main sources of tubulin inhibitors. However, due to the variety of compound structure types, it has always been difficult for researchers to screen out polymerization inhibitors with simple operation, high efficiency and low cost. A large number of articles have reported the screening methods of tubulin inhibitors and their biological activity. In this article, the biological activity detection methods of tubulin polymerization inhibitors are reviewed. Thus, it provides a theoretical basis for the further study of tubulin polymerization inhibitors and the selection of methods for tubulin inhibitors.

Mechanism of the O2 (1 Δg ) generation from the Cl2 /H2 O2 basic aqueous solution explored by the combined ab initio calculation and nonadiabatic dynamics simulation.

In the present work, mechanism of the O2 (1 Δg ) generation from the reaction of the dissolved Cl2 with H2 O2 in basic aqueous solution has been explored by the combined ab initio calculation and nonadiabatic dynamics simulation, together with different solvent models. Three possible pathways have been determined for the O2 (1 Δg ) generation, but two of them are sequentially downhill processes until formation of the OOCl- complex with water, which are of high exothermic character. Once the complex is formed, singlet molecular oxygen is easily generated by its decomposition along the singlet-state pathway. However, triplet molecular oxygen of O2 ( Σ 3 g - ) can be produced with considerable probability through nonadiabatic intersystem crossing in the 1 Δg / Σ 3 g - intersection region. It has been found that the coupled solvent, heavy-atom, and nonadiabatic effects have an important influence on the quantum yield of the O2 (1 Δg ) generation. © 2018 Wiley Periodicals, Inc.

Targeting ALDH2 with disulfiram/copper reverses the resistance of cancer cells to microtubule inhibitors.

Disulfiram (DSF) in combination with copper (Cu) has been reported to override drug resistance in cancer cells, and DSF combined with chemotherapy based on the microtubule inhibitor vinorelbine appears to prolong survival in non-small cell lung cancer patients. Here, we investigated the mechanisms underlying these findings. DSF/Cu reversed the microtubule inhibitor resistance in A549/Taxol and KB/VCR cells in vitro, and had anti-tumor effects in A549/Taxol and KB/VCR xenograft mice. DSF/Cu and DSF reduced the cancer stem cell (CSC) characteristics of drug-resistant A549/Taxol and KB/VCR cells, including sphere formation, colony generation and migration, and DSF/Cu was more effective than DSF alone. DSF/Cu also decreased the aldehyde dehydrogenase (ALDH) activity and the expression of P-gp and stem cell transcription factors in A549/Taxol and KB/VCR cells. Knockdown of ALDH2 attenuated the CSC characteristics of resistant cancer cells and enhanced their sensitivity to Taxol or VCR. Importantly, DSF/Cu treatment inhibited the expression of ALDH2 in vitro and in vivo. Our findings suggest that DSF/Cu reverses microtubule inhibitor resistance in cancer cells by suppressing ALDH2 expression, and Cu improves the activity of DSF.

Aberrant expressed long non-coding RNAs in laryngeal squamous-cell carcinoma.

PURPOSE: Laryngeal squamous-cell carcinoma (LSCC) is the second most common malignant tumor of head and neck squamous cell carcinoma. The study was aimed to identify key long non-coding RNAs (lncRNAs) biomarkers for LSCC. METHODS: Differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) between LSCC and adjacent tissues were obtained based on The Cancer Genome Atlas. DElncRNA-DEmRNAs co-expression and DElncRNA-nearby-target DEmRNA interaction networks were constructed. Receiver operating characteristic and survival analysis were performed. A published dataset were as used to validate the result of bioinformatics analysis. RESULTS: We obtained 1103 DEmRNAs and 306 DElncRNAs between LSCC and adjacent tissues. A total of 338 DElncRNA-DEmRNA co-expression pairs and 229 DElncRNA-nearby-target DEmRNA pairs were obtained. Ten DElncRNAs and six DEmRNAs has great diagnostic value for LSCC. HOXB9 has potential prognostic value for LSCC. The results of GSE84957 validation were generally consistent with our results. CONCLUSION: Our study provided clues for understanding the mechanism and developing potential biomarkers for LSCC.

Disulfiram combined with copper inhibits metastasis and epithelial-mesenchymal transition in hepatocellular carcinoma through the NF-κB and TGF-ß pathways.

Late-stage hepatocellular carcinoma (HCC) usually has a low survival rate because of the high risk of metastases and the lack of an effective cure. Disulfiram (DSF) has copper (Cu)-dependent anticancer properties in vitro and in vivo. The present work aims to explore the anti-metastasis effects and molecular mechanisms of DSF/Cu on HCC cells both in vitro and in vivo. The results showed that DSF inhibited the proliferation, migration and invasion of HCC cells. Cu improved the anti-metastatic activity of DSF, while Cu alone had no effect. Furthermore, DSF/Cu inhibited both NF-κB and TGF-ß signalling, including the nuclear translocation of NF-κB subunits and the expression of Smad4, leading to down-regulation of Snail and Slug, which contributed to phenotype epithelial-mesenchymal transition (EMT). Finally, DSF/Cu inhibited the lung metastasis of Hep3B cells not only in a subcutaneous tumour model but also in an orthotopic liver metastasis assay. These results indicated that DSF/Cu suppressed the metastasis and EMT of hepatic carcinoma through NF-κB and TGF-ß signalling. Our study indicates the potential of DSF/Cu for therapeutic use.

The SWI/SNF Chromatin Regulator BRG1 Modulates the Transcriptional Regulatory Activity of the Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1.

During the lytic phase of Epstein-Barr virus (EBV), binding of the transactivator Zta to the origin of lytic replication (oriLyt) and the BHLF1 transcript, forming a stable RNA-DNA hybrid, is required to initiate viral DNA replication. EBV-encoded viral DNA replication proteins form complexes to amplify viral DNA. BMRF1, the viral DNA polymerase accessory factor, is essential for lytic DNA replication and also known as a transcriptional regulator of the expression of BHLF1 and BALF2 (single-stranded DNA [ssDNA]-binding protein). In order to determine systematically how BMRF1 regulates viral transcription, a BMRF1 knockout bacmid was generated to analyze viral gene expression using a viral DNA microarray. We found that a subset of Rta-responsive late genes, including BcLF1, BLLF1, BLLF2, and BDLF3, were downregulated in cells harboring a BMRF1 knockout EBV bacmid (p2089ΔBMRF1). In reporter assays, BMRF1 appears to transactivate a subset of viral late promoters through distinct pathways. BMRF1 activates the BDLF3 promoter in an SP1-dependent manner. Notably, BMRF1 associates with the transcriptional regulator BRG1 in EBV-reactivated cells. BMRF1-mediated transactivation activities on the BcLF1 and BLLF1 promoters were attenuated by knockdown of BRG1. In BRG1-depleted EBV-reactivated cells, BcLF1 and BLLF1 transcripts were reduced in number, resulting in reduced virion secretion. BMRF1 and BRG1 bound to the adjacent upstream regions of the BcLF1 and BLLF1 promoters, and depletion of BRG1 attenuated the recruitment of BMRF1 onto both promoters, suggesting that BRG1 is involved in BMRF1-mediated regulation of these two genes. Overall, we reveal a novel pathway by which BMRF1 can regulate viral promoters through interaction with BRG1.IMPORTANCE The cascade of viral gene expression during Epstein-Barr virus (EBV) replication is exquisitely regulated by the coordination of the viral DNA replication machinery and cellular factors. Upon lytic replication, the EBV immediate early proteins Zta and Rta turn on the expression of early proteins that assemble into viral DNA replication complexes. The DNA polymerase accessory factor, BMRF1, also is known to transactivate early gene expression through its interaction with SP1 or Zta on specific promoters. Through a global analysis, we demonstrate that BMRF1 also turns on a subset of Rta-regulated, late structural gene promoters. Searching for BMRF1-interacting cellular partners revealed that the SWI/SNF chromatin modifier BRG1 contributes to BMRF1-mediated transactivation of a subset of late promoters through protein-protein interaction and viral chromatin binding. Our findings indicate that BMRF1 regulates the expression of more viral genes than thought previously through distinct viral DNA replication-independent mechanisms.

Theoretical investigations on diamondoids (CnHm, n = 10-41): Nomenclature, structural stabilities, and gap distributions.

Combining the congruence check and the first-principles calculations, we have systematically investigated the structural stabilities and gap distributions of possible diamondoids (CnHm) with the carbon numbers (n) from 10 to 41. A simple method for the nomenclature is proposed, which can be used to distinguish and screen the candidates with high efficiency. Different from previous theoretical studies, the possible diamondoids can be enumerated according to our nomenclature, without any pre-determination from experiments. The structural stabilities and electronic properties have been studied by density functional based tight binding and first-principles methods, where a nearly linear correlation is found between the energy gaps obtained by these two methods. According to the formation energy of structures, we have determined the stable configurations as a function of chemical potential. The maximum and minimum energy gaps are found to be dominated by the shape of diamondoids for clusters with a given number of carbon atoms, while the gap decreases in general as the size increases due to the quantum confinement.

Photochromic Mechanism of a Bridged Diarylethene: Combined Electronic Structure Calculations and Nonadiabatic Dynamics Simulations.

Intramolecularly bridged diarylethenes exhibit improved photocyclization quantum yields because the anti-syn isomerization that originally suppresses photocyclization in classical diarylethenes is blocked. Experimentally, three possible channels have been proposed to interpret experimental observation, but many details of photochromic mechanism remain ambiguous. In this work we have employed a series of electronic structure methods (OM2/MRCI, DFT, TDDFT, RI-CC2, DFT/MRCI, and CASPT2) to comprehensively study excited state properties, photocyclization, and photoreversion dynamics of 1,2-dicyano[2,2]metacyclophan-1-ene. On the basis of optimized stationary points and minimum-energy conical intersections, we have refined experimentally proposed photochromic mechanism. Only an S1/S0 minimum-energy conical intersection is located; thus, we can exclude the third channel experimentally proposed. In addition, we find that both photocyclization and photoreversion processes use the same S1/S0 conical intersection to decay the S1 system to the S0 state, so we can unify the remaining two channels into one. These new insights are verified by our OM2/MRCI nonadiabatic dynamics simulations. The S1 excited-state lifetimes of photocyclization and photoreversion are estimated to be 349 and 453 fs, respectively, which are close to experimentally measured values: 240 ± 60 and 250 fs in acetonitrile solution. The present study not only interprets experimental observations and refines previously proposed mechanism but also provides new physical insights that are valuable for future experiments.

Antiinfective therapy with a small molecule inhibitor of Staphylococcus aureus sortase.

Methicillin-resistant Staphylococcus aureus (MRSA) is the most frequent cause of hospital-acquired infection, which manifests as surgical site infections, bacteremia, and sepsis. Due to drug-resistance, prophylaxis of MRSA infection with antibiotics frequently fails or incites nosocomial diseases such as Clostridium difficile infection. Sortase A is a transpeptidase that anchors surface proteins in the envelope of S. aureus, and sortase mutants are unable to cause bacteremia or sepsis in mice. Here we used virtual screening and optimization of inhibitor structure to identify 3-(4-pyridinyl)-6-(2-sodiumsulfonatephenyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole and related compounds, which block sortase activity in vitro and in vivo. Sortase inhibitors do not affect in vitro staphylococcal growth yet protect mice against lethal S. aureus bacteremia. Thus, sortase inhibitors may be useful as antiinfective therapy to prevent hospital-acquired S. aureus infection in high-risk patients without the side effects of antibiotics.