Identification of Toxic Proteins Encoded by Mycobacteriophage TM4 Using a Next-Generation Sequencing-Based Method.

Mycobacteriophages are viruses that specifically infect mycobacteria and which, due to their diversity, represent a large gene pool. Characterization of the function of these genes should provide useful insights into host-phage interactions. Here, we describe a next-generation sequencing (NGS)-based, high-throughput screening approach for the identification of mycobacteriophage-encoded proteins that are toxic to mycobacteria. A plasmid-derived library representing the mycobacteriophage TM4 genome was constructed and transformed into Mycobacterium smegmatis. NGS and growth assays showed that the expression of TM4 gp43, gp77, -78, and -79, or gp85 was toxic to M. smegmatis. Although the genes associated with bacterial toxicity were expressed during phage infection, they were not required for lytic replication of mycobacteriophage TM4. In conclusion, we describe here an NGS-based approach which required significantly less time and resources than traditional methods and allowed the identification of novel mycobacteriophage gene products that are toxic to mycobacteria. IMPORTANCE The wide spread of drug-resistant Mycobacterium tuberculosis has brought an urgent need for new drug development. Mycobacteriophages are natural killers of M. tuberculosis, and their toxic gene products might provide potential anti-M. tuberculosis candidates. However, the enormous genetic diversity of mycobacteriophages poses challenges for the identification of these genes. Here, we used a simple and convenient screening method, based on next-generation sequencing, to identify mycobacteriophage genes encoding toxic products for mycobacteria. Using this approach, we screened and validated several toxic products encoded by mycobacteriophage TM4. In addition, we also found that the genes encoding these toxic products are nonessential for lytic replication of TM4. Our work describes a promising method for the identification of phage genes that encode proteins that are toxic to mycobacteria and which might facilitate the identification of novel antimicrobial molecules.

Application of combined CRISPR screening for genetic and chemical-genetic interaction profiling in Mycobacterium tuberculosis.

CRISPR screening, including CRISPR interference (CRISPRi) and CRISPR-knockout (CRISPR-KO) screening, has become a powerful technology in the genetic screening of eukaryotes. In contrast with eukaryotes, CRISPR-KO screening has not yet been applied to functional genomics studies in bacteria. Here, we constructed genome-scale CRISPR-KO and also CRISPRi libraries in Mycobacterium tuberculosis (Mtb). We first examined these libraries to identify genes essential for Mtb viability. Subsequent screening identified dozens of genes associated with resistance/susceptibility to the antitubercular drug bedaquiline (BDQ). Genetic and chemical validation of the screening results suggested that it provided a valuable resource to investigate mechanisms of action underlying the effects of BDQ and to identify chemical-genetic synergies that can be used to optimize tuberculosis therapy. In summary, our results demonstrate the potential for efficient genome-wide CRISPR-KO screening in bacteria and establish a combined CRISPR screening approach for high-throughput investigation of genetic and chemical-genetic interactions in Mtb.

Toxin-Antitoxin Systems Alter Adaptation of Mycobacterium smegmatis to Environmental Stress.

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in prokaryotes, but their biological importance is poorly understood. Mycobacterium smegmatis contains eight putative TA systems. Previously, seven TAs have been studied, with five of them being verified as functional. Here, we show that Ms0251-0252 is a novel TA system in that expression of the toxin Ms0251 leads to growth inhibition that can be rescued by the antitoxin Ms0252. To investigate the functional roles of TA systems in M. smegmatis, we deleted the eight putative TA loci and assayed the mutants for resistance to various stresses. Deletion of all eight TA loci resulted in decreased survival under starvation conditions and altered fitness when exposed to environmental stresses. Furthermore, we showed that deletion of the eight TA loci decreased resistance to phage infection in Sauton medium compared with the results using 7H10 medium, suggesting that TA systems might have different contributions depending on the nutrient environment. Furthermore, we found that MazEF specifically played a dominant role in resistance to phage infection. Finally, transcriptome analysis revealed that MazEF overexpression led to differential expression of multiple genes, including those related to iron acquisition. Altogether, we demonstrate that TA systems coordinately function to allow M. smegmatis to adapt to changing environmental conditions. IMPORTANCE Toxin-antitoxin (TA) systems are mechanisms for rapid adaptation of bacteria to environmental changes. Mycobacterium smegmatis, a model bacterium for studying Mycobacterium tuberculosis, encodes eight putative TA systems. Here, we constructed an M. smegmatis mutant with deletions of all eight TA-encoding genes and evaluated the resistance of these mutants to environmental stresses. Our results showed that different TA systems have overlapping and, in some cases, opposing functions in adaptation to various stresses. We suggest that complementary TA modules may function together to regulate the bacterial stress response, enabling adaptation to changing environments. Together, this study provides key insights into the roles of TA systems in resistance to various environmental stresses, drug tolerance, and defense against phage infection.

Sex-related differences in the efficacy of immune checkpoint inhibitors in malignancy: a systematic review and meta-analysis.

Although disease susceptibility is known to differ between men and women, it is controversial whether the efficacy of immune checkpoint inhibitors for malignancies also differs between the sexes. We conducted a meta-analysis to explore the impact of sex on immune checkpoint inhibitor treatment outcomes. We searched PubMed, Embase and the Cochrane Library databases from inception to October 1, 2020 for randomized controlled trials of immune checkpoint inhibitors with hazard ratios (HRs) stratified by sex. We calculated the pooled HRs for men and women using the ln(HR), and assessed the heterogeneity between the two estimates through an interaction test. In total, 22,268 patients from 39 randomized controlled trials were included. Immune checkpoint inhibitors yielded better overall survival than conventional agents in both men (HR: 0.75, 95% confidence interval [CI]: 0.71-0.80) and women (HR: 0.77, 95% CI: 0.70-0.85). Progression-free survival benefits were also observed in both men (HR: 0.64, 95% CI: 0.58-0.70) and women (HR: 0.67, 95% CI: 0.58-0.77) treated with immune checkpoint inhibitors. No sex differences in the response to immune checkpoint inhibitors were found when overall survival and progression-free survival were used as the endpoints.

Does fully-threaded Headless Compression Screw provide a length-stable fixation in undisplaced femoral neck fractures?

OBJECTIVE: Femoral neck shortening is a common complication after surgical treatment for intracapsular femoral neck fractures. This study investigated whether fully-threaded Headless Compression Screw (FTHCS) can be a more length-stable implant than partially-threaded cannulated screw (PTCS) in reducing femoral neck shortening. METHODS: A total of 50 patients with undisplaced femoral neck fractures (17 treated by internal fixation with three FTHCS, and 33 treated by three PTCS) from 2011 to 2014 were enrolled in this study. The radiography of the hips and medical records were reviewed for proximal femur geometry and complications. RESULTS: Significant shortening of the femoral neck length until union were noted in both group (FTHCS group: -2.5 mm, p = 0.045; PTCS group: -2.4 mm, p = 0.011). There was no significant difference in the length of femoral neck shortening between groups (p = 0.855). Age was the only significant risk factor for >5 mm of femoral neck shortening (p = 0.041). The femoral neck-shaft angle tended to reduce and become more varus in both groups (FTHCS: -2.7°, SD = 4.5, p = 0.028; PTCS: -5.0°, SD = 8.3, p = 0.002), but the differences between groups were nonsignificant (p = 0.577). The complication rates were similar between FTHCS and PTCS (17.6% and 21.2%, p > 0.999). CONCLUSION: The FTHCSs may be a substitute for PTCSs, but it cannot prevent femoral neck shortening and varus collapse after fracture fixation. Future studies should focus on how to preserve femoral neck length and hip function after femoral neck fractures.

Creation and Transfer of Coherence via Technique of Stimulated Raman Adiabatic Passage in Triple Quantum Dots.

We propose a scheme for creation and transfer of coherence among ground state and indirect exciton states of triple quantum dots via the technique of stimulated Raman adiabatic passage. Compared with the traditional stimulated Raman adiabatic passage, the Stokes laser pulse is replaced by the tunneling pulse, which can be controlled by the externally applied voltages. By varying the amplitudes and sequences of the pump and tunneling pulses, a complete coherence transfer or an equal coherence distribution among multiple states can be obtained. The investigations can provide further insight for the experimental development of controllable coherence transfer in semiconductor structure and may have potential applications in quantum information processing.

A Systematic Review of Chiari I Malformation: Techniques and Outcomes.

OBJECTIVE: To elucidate the most efficacious treatments of Chiari I malformation (CIM). METHODS: A literature search was performed using PubMed, CINAHL/Ovid, Cochrane library, and the Elsevier database. The key words "Chiari I malformation," "Chiari malformation type I," "surgery," and "treatment" were used for the search. Articles had to be peer reviewed and provide primary outcomes measured by clinical and radiographic outcomes after surgical treatments. Exclusion criteria included non-English-language articles, case reports, commentaries, information from textbooks and expert opinions, and articles that did not provide outcomes concerning specific surgical methods. Patients included were classified into 4 groups according the procedure: only bony decompression but not duroplasty (group I), bony decompression plus duroplasty (group II), bony decompression plus the resection of tonsils (group III), and shunt (group IV). RESULTS: Eighteen studies were identified. Groups II and III had a significantly higher improvement rate (82.25%, 86.10%, P < 0.05) of outcomes with regard to clinical signs and symptoms than the other groups. Group IV showed a statistically higher rate (30.49%, P < 0.05) of aggregating clinical signs and symptoms. In patients with syringomyelia, group III showed better clinical improvement (96.08%). Group II displayed a significantly higher rate of decrease in the size of cavities (83.33%, P < 0.05). Group IV had a statistically higher rate of increase in the size of cavities (33.87%, P < 0.05). CONCLUSIONS: Only bony decompression cannot achieve satisfactory outcomes. Bony decompression plus duroplasty showed the most favorable outcomes. Resection of tonsils was not recommended because of the high rate of side effects. Shunt may aggregate clinical signs and symptoms and increase the size of cavities.

Recent progress in HER2 associated breast cancer.

Breast cancer is the most common cancer worldwide among women and the second most common cancer. Approximately 15-23% of breast cancers over-express human epidermal growth factor receptor2 (HER2), a 185-kDa transmembrane tyrosine kinase, which is mainly found at the cell surface of tumor cells. HER2-positive breast cancer, featuring amplification of HER2/neu and negative expression of ER and PR, has the three following characteristics: rapid tumor growth, lower survival rate, and better response to adjuvant therapies. Clinically, it is notable for its role in a pathogenesis that is associated with increased disease recurrence and acts as a worse prognosis. At the same time, it represents a good target for anti-cancer immunotherapy despite the prevalence of drug resistance. New treatments are a major topic of research, and a brighter future can be expected. This review discusses the role of HER2 in breast cancer, therapeutic modalities available and prognostic factors.

Enhanced waveguide-type ultraviolet electroluminescence from ZnO/MgZnO core/shell nanorod array light-emitting diodes via coupling with Ag nanoparticles localized surface plasmons.

Localized surface plasmon (LSP) enhanced waveguide-type ultraviolet light-emitting diodes (LEDs) were fabricated by sputtering Ag nanoparticles (Ag-NPs) onto ZnO/MgZnO core/shell nanorod array (CS-NRA)/p-GaN heterostructures. A ∼9-fold enhancement of ZnO ultraviolet electroluminescence (EL) was demonstrated by the Ag-NPs decorated LED compared with the device without Ag-NPs. Angle-dependent EL measurements, as well as finite-difference time-domain simulations of the EL intensity spatial distribution, confirmed the waveguide-type EL transmission mode along the NR's axial direction. The increased spontaneous emission rate observed in time-resolved spectroscopy suggested that the ZnO EL enhancement was attributed to LSP-exciton/polariton coupling. However, a direct coupling is very difficult to achieve between Ag-LSPs and electron-hole pairs in the active region due to their "remote" separation. Thereby, two possible models involving the dynamic process of interactions among excitons, photons, and LSPs, were established to understand the selective enhancement of ZnO EL.

Mitogen-activated protein kinase signal transduction in solid tumors.

Mitogen-activated protein kinase (MAPK) is an important signaling pathway in living beings in response to extracellular stimuli. There are 5 main subgroups manipulating by a set of sequential actions: ERK(ERK1/ ERK2), c-Jun N(JNK/SAPK), p38 MAPK(p38α, p38ß, p38γ and p38δ), and ERK3/ ERK4/ ERK5. When stimulated, factors of upstream or downstream change, and by interacting with each other, these groups have long been recognized to be related to multiple biologic processes such as cell proliferation, differentiation, death, migration, invasion and inflammation. However, once abnormally activated, cancer may occur. Several components of the MAPK network have already been proposed as targets in cancer therapy, such as p38, JNK, ERK, MEK, RAF, RAS, and DUSP1. Among them, alteration of the RAS-RAF-MEK-ERK-MAPK(RAS-MAPK) pathway has frequently been reported in human cancer as a result of abnormal activation of receptor tyrosine kinases or gain-of-function mutations in genes. The reported roles of MAPK signaling in apoptotic cell death are controversial, so that further in-depth investigations are needed to address these controversies. Based on an extensive analysis of published data, the goal of this review is to provide an overview on recent studies about the mechanism of MAP kinases, and how it generates certain tumors, as well as related treatments.

Enhanced ultraviolet emission and improved spatial distribution uniformity of ZnO nanorod array light-emitting diodes via Ag nanoparticles decoration.

Localized surface plasmon (LSP) enhanced ultraviolet (UV) light-emitting diodes (LEDs) were fabricated by embedding a ZnO nanorod array/p-GaN film heterostructure into a Ag-nanoparticles/PMMA composite. By optimizing the concentration of Ag nanoparticles in PMMA, two distinct changes in electroluminescence (EL) spectra were observed: (1) the UV EL component from ZnO excitons was selectively enhanced more than 13-fold and the entire spectral lineshape was changed and (2) the spatial uniformity of the output photon intensity was improved and the linewidth of an angular distribution curve was increased by ∼2 times. These observations can be attributed to near-field optical coupling between Ag LSPs and ZnO excitons. Time-resolved luminescence measurements and a model calculation reveal that the optical coupling results in the increase of the spontaneous emission rate and internal quantum efficiency of Ag-nanoparticles-decorated ZnO nanorod arrays. Moreover, the LSP-exciton interaction allows the device's EL to be coupled out of the nanorod waveguide and to be isotropically scattered into every direction, thus broadening the angular distribution of the EL intensity.

Observation of the fluorescence spectrum for a driven cascade model system in atomic beam.

We experimentally study the resonance fluorescence from an excited two-level atom when the atomic upper level is coupled by a nonresonant field to a higher-lying state in a rubidium atomic beam. The heights, widths and positions of the fluorescence peaks can be controlled by modifying the detuning of the auxiliary field. We explain the observed spectrum with the transition properties of the dressed states generated by the coupling of the two laser fields. We also attribute the line narrowing to the effects of Spontaneously Generated Coherence between the close-lying levels in the dressed state picture generated by the auxiliary field. And the corresponding spectrum can be viewed as the evidence of Spontaneously Generated Coherence. The experimental results agree well with calculations based on the density-matrix equations.

Control of spontaneous emission from a micro-wave driven atomic system.

We demonstrate coherent control of spontaneous emission from an indirectly coupled transition in a microwave driven four-level atomic system. The transition of concern is not directly coupled by any laser fields, while the ground state is coupled to another ground state by a microwave field. We show that the coupling of the microwave field produces interesting features such as double narrow lines in the emission spectrum. The heights, widths and positions of the emission peaks can be controlled by modifying the Rabi frequency and detuning of the microwave field. We discuss the spectra in the dressed states basis.

[Expression of HER2/neu in meningiomas: an immunohistochemistry and fluorescence in situ hybridization study].

OBJECTIVE: To investigate the expression of HER2/neu, Ki-67 and TK1 protein in meningiomas in correlation with tumor grades and recurrence. METHODS: Twenty cases of each of the following types of meningiomas were selected for the study, namely: the benign non-recurrent, recurrent benign, atypical and malignant, according to the World Health Organization (WHO) histological classification of nervous system, 2007. Immunohistochemistry study for HER2/neu, Ki-67 and TK1 protein was performed. HER2/neu gene amplification was detected using FISH. Cases with HER2 protein overexpression were studied by immunohistochemistry staining. The results of the biomarker assays were also used to study the correlationship with the tumor grades and tumor recurrency. RESULTS: Immunohistochemistry showed that the positive rates of HER2 expression in non-recurrence benign group, recurrence benign group, atypical group and malignant group were 3 cases (15%), 6 cases (30%), 7 cases (35%), and 10 cases (50%), respectively (P < 0.05). A higher tumor grade was correlated with a higher rate of HER2/neu expression. The Ki-67 and TK1 labeling index (LI) in non-recurrence group were lower than those in the atypical or malignant group (P < 0.05), whereas the atypical group had lower LI than that of the malignant group (P < 0.05). Higher levels of LI of Ki-67 and TK1 were correlated with higher tumor grades and recurrence of the benign meningiomas (P < 0.05). Expression of HER2 was positively correlated with Ki-67 and TK1 (r = 0.445, P < 0.01; r = 0.501, P < 0.01, respectively), and there was a positive correlation between Ki-67 and TK1 (r = 0.450, P < 0.01) as well. HER2/neu gene copy amplification in 7 of 26 cases (26.9%) of HER2 immunopositive meningiomas. The rates of HER2/neu gene amplification were 0 in tumors with 1+ immunopositivity, 4/6 in tumor with 2+ immunopositivity and 3/4 in tumor with 3+ immunopositivity. HER2/neu gene amplification in 3+ and 2+ immunopositive cases had no statistical significance (P > 0.05). Aneuploidy of chromosome 17 existed in 9 of 26 of HER2 immunopositive meningiomas (34.6%). However, the rates of chromosome 17 aneuploidy had no significant difference among tumors with variable HER2/neu imumopositivity (P > 0.05). CONCLUSIONS: High levels of HER2 and Ki-67 or TK1 expression correlate with the increase of tumor grades and tumor recurrence. HER2/neu gene amplification is seen in a subset of meningiomas with the protein expression (26.9%). A combination of biomarker study including HER2/neu, Ki-67 and TK1 may be useful in predicting the biological behavior of meningiomas.

Investigation of spontaneously generated coherence in dressed states of (85)Rb atoms.

We investigate the effects of spontaneously generated coherence (SGC) on the electromagnetically induced transparency features in a four-level Lambda-type system. We show that double-transparency windows and a controllable narrow absorption peak can be obtained with the presence of SGC. We present an equivalent system without the rigorous requirement of close-lying levels to observe the phenomena. We also experimentally demonstrate the corresponding features in a rubidium atomic beam.