An evidence-based guideline on treating lumbar disc herniation with traditional Chinese medicine.

BACKGROUND: Lumbar disc herniation (LDH), as one of the most common causes of lower back pain, imposes a heavy economic burden on patients and society. Conservative management is the first-line choice for the majority of LDH patients. Traditional Chinese medicine (TCM) is an important part of conservative treatment and has attracted more and more international attention. STUDY DESIGN: Evidence-based guideline. METHODS: We formed a guideline panel of multidisciplinary experts. The clinical questions were identified on the basis of a systematic literature search and a consensus meeting. We searched the literature for direct evidence on the management of LDH and assessed its certainty-generated recommendations using the grading of recommendations, assessment, development, and evaluation (GRADE) approach. RESULTS: The guideline panel made 20 recommendations, which covered the use of Shentong Zhuyu decoction, Shenzhuo decoction, Simiao San decoction, Duhuo Jisheng decoction, Yaobitong capsule, Yaotongning capsule, Osteoking, manual therapy, needle knife, manual acupuncture, electroacupuncture, Chinese exercise techniques (Tai Chi, Baduanjin, or Yijinjing), and integrative medicine, such as combined non-steroidal anti-inflammatory drugs, neural nutrition, and traction. Recommendations were either strong or weak, or in the form of ungraded consensus-based statement. CONCLUSION: This is the first LDH treatment guideline for TCM and integrative medicine with a systematic search, synthesis of evidence, and using the GRADE method to rate the quality of evidence. We hope these recommendations can help support healthcare workers caring for LDH patients.

[Salvianolic acid A contributes to cartilage endplate cell restoration by regulating miR-940 and miR-576-5p].

OBJECTIVE: To investigate whether Salvianolic acid A (SAA) can restore cartilage endplate cell degeneration of intervertebral discs and to identify the mechanism via regulation of micro-RNA. METHODS: Cartilage endplate cells were isolated from lumbar intervertebral disc surgical samples and were treated with serum containing a series of concentrations of SAA (2, 5, and 10 ?M) for 24, 48, and 72 h to identify a proper dose and treatment time of SAA. The effect SAA on interlenkin-1ß (IL-1ß)-induced extracellular matrix degradation of cartilage endplate cells were analyzed by Alcian blue staining and assessment of the expression levels of ADAMTS-5, MMP3 and Col2a1. Further, the potential target miRNAs were preliminarily screened by micro-RNA sequencing combining qRT-PCR and Western blot, and then, the miRNAs mimics and inhibitors were used to verify the regulatory effect of SAA on potential target miRNAs. RESULTS: The 10 µM SAA treatment for 48 h significantly enhanced the viability of cartilage endplate cells, and increased Col2a1 expression and glycosaminoglycan accumulation that were repressed by IL-1ß, and reduced the effect of IL-1ß on ADAMTS-5, and MMP3. Screening analysis based on micro-RNA sequencing and Venny analysis identified the downstream micro-RNAs, including miR-940 and miR-576-5p. Then, the miR-940-mimic or miR-576-5p-mimic were transfected into CEPCs. Compared with the SAA group, the expression of ADAMTS-5 and MMP3 increased significantly and the expression of COL2A1 obviously decreased after overexpression of miR-940 or miR-576-5p in CEPCs. CONCLUSION: Salvianolic acid A attenuated the IL-1ß-induced extracellular matrix degradation of cartilage endplate cells by targeting regulate the miR-940 and the miR-576-5p.

The diagnostic value of circulating abnormal cells in early lung cancer.

Lung cancer is the leading cause of cancer-related deaths globally. Early detection of lung cancer can lead to more effective treatment and improved survival. Circulatory abnormal cells (CACs) with specific chromosomal variation may be used to diagnose lung cancer and to differentiate benign from malignant nodules. The value of CAC in precancer diagnosis, however, remains controversial. In this study, a systematic review and meta-analysis are conducted to clarify the diagnostic value of CAC in early-stage lung cancer. A systematic literature search was conducted using the following medical topic title terms and text-free words: "circulating genetically abnormal cells", "CACs", "liquid biopsy", "early lung cancer", "non-small cell lung cancer", "diagnostic accuracy", "sensitivity" and "specificity" in Science Direct, CNKI and Wanfang databases, respectively. Sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and area under the curve were analyzed by STATA15.0 (MP) software. Deek funnel plots were used to assess potential publication bias. Heterogeneity was tested using the I2 statistic and the Cochrane Q test. 7 major studies were included in this meta-analysis, and a total of 53728 participants were analyzed. In the diagnosis of early lung cancer, CAC had pooled sensitivity, specificity, and receiver operating characteristics of 0.80 (95% CI: 0.73-0.86), 0.85 (95% CI: 0.69-0.94), and 0.87 (95% CI: 0.84-0.90). The combined positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and diagnostic score were 23.36 (95% CI: 7.33-74.46), 5.42 (95% CI: 2.37-12.43), 0.23 (95% CI: 0.16-0.35) and 3.15 (95% CI: 1.99-4.31) respectively. Publication bias was not detected. The CAC is effective at detecting lung cancer in its early stages.

Application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer.

Colon cancer is the fourth leading cause of cancer death worldwide, and its progression is accompanied by a complex array of genetic variations. CRISPR/Cas9 can identify new drug-resistant or sensitive mutations in colon cancer, and can use gene editing technology to develop new therapeutic targets and provide personalized treatments, thereby significantly improving the treatment of colon cancer patients. CRISPR/Cas9 systems are driving advances in biotechnology. RNA-directed Cas enzymes have accelerated the pace of basic research and led to clinical breakthroughs. This article reviews the rapid development of CRISPR/Cas in colon cancer, from gene editing to transcription regulation, gene knockout, genome-wide CRISPR tools, therapeutic targets, stem cell genomics, immunotherapy, metabolism-related genes and inflammatory bowel disease. In addition, the limitations and future development of CRISPR/Cas9 in colon cancer studies are reviewed. In conclusion, this article reviews the application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer.

ZG16 promotes T-cell mediated immunity through direct binding to PD-L1 in colon cancer.

Immunotherapy using programmed cell death 1 (PD1) inhibitors has shown great efficacy in colorectal cancer patients harboring mismatch-repair-deficient (dMMR) and microsatellite instability-high (MSI-H) alterations. We previously showed a negative correlation of zymogen granule protein 16 (ZG16) with programmed death-ligand 1 (PD-L1) expression in patients with colorectal cancer. However, how ZG16 regulates PD-L1 expression is unclear. In this study, we showed that ZG16 can directly bind to glycosylated PD-L1 through its lectin domain, leading to PD-L1 degradation. Mutations on the lectin domain of ZG16 largely inhibit the interaction between ZG16 and PD-L1. Importantly, ZG16 overexpression suppressed tumor growth in two syngeneic mouse models through blockage of PD-L1 expression in cancer cells meanwhile suppression of PD1 expression in T cells. We also showed that ZG16 could improve the effect of chemotherapy and may be delivered as a protein to serve as an immune checkpoint inhibitor to promote T-cell mediated immunity.

Observation of novel COX20 mutations related to autosomal recessive axonal neuropathy and static encephalopathy.

Cytochrome c oxidase 20 (COX20)/FAM36A encodes a conserved protein that is important for the assembly of COX, complex IV of the mitochondrial respiratory chain. A homozygous mutation (p.Thr52Pro) in COX20 gene has been previously described to cause muscle hypotonia and ataxia. In this study, we describe two patients from a non-consanguineous family exhibiting autosomal recessive sensory-dominant axonal neuropathy and static encephalopathy. The whole-exome sequencing analysis revealed that both patients harbored compound heterozygous mutations (p.Lys14Arg and p.Trp74Cys) of COX20 gene. The pathogenicity of the variants was further supported by morphological alternations of mitochondria observed in sural nerve and decreased COX20 protein level of peripheral blood leucocytes derived from the patients. In conclusion, COX20 might be considered as a candidate gene for the complex inherited disease. This observation broadens the clinical and genetic spectrum of COX20-related disease. However, due to the limitation of a single-family study, additional cases and studies are definitely needed to further confirm the association.

PARP1 depletion improves mitochondrial and heart function in Chagas disease: Effects on POLG dependent mtDNA maintenance.

Chagasic cardiomyopathy is caused by Trypanosoma cruzi infection. Poly(ADP-ribose) polymerase 1 (PARP1) is known for its function in nuclear DNA repair. In this study, we have employed genetic deletion and chemical inhibition approaches to determine the role of PARP1 in maintaining mtDNA dependent mitochondrial function in Chagas disease. Our data show that expression of PARP1 and protein PARylation were increased by >2-fold and >16-fold, respectively, in the cytosolic, nuclear, and mitochondrial fractions of the human cardiac myocytes and the myocardium of wildtype (WT) mice chronically infected with T. cruzi. The nuclear and cytosolic PARP1/PAR did not interfere with the transcription and translation of the components of the mtDNA replisome machinery in infected cardiomyocytes and chagasic murine myocardium. However, PARP1 binding to Polymerase γ and mtDNA in mitochondria were increased, and associated with a loss in mtDNA content, mtDNA-encoded gene expression, and oxidative phosphorylation (OXPHOS) capacity, and an increase in mitochondrial ROS production in cells and heart of WT mice infected with T. cruzi. Subsequently, an increase in oxidative stress, and cardiac collagen deposition, and a decline in LV function was noted in chagasic mice. Genetic deletion of PARP1 or treatment with selective inhibitor of PARP1 (PJ34) improved the mtDNA content, mitochondrial function, and oxidant/antioxidant balance in human cardiomyocytes and chronically infected mice. Further, PARP1 inhibition was beneficial in preserving the cardiac structure and left ventricular function in chagasic mice. We conclude that PARP1 overexpression is associated with a decline in Pol γ-dependent maintenance of mtDNA content, mtDNA-encoded gene expression, and mitochondrial respiratory function, and subsequently contributes to an increase in mtROS and oxidative stress in chagasic myocardium. Inhibition of mitochondrial PARP1/PAR offers a novel therapy in preserving the mitochondrial and LV function in chronic Chagas disease.

Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based.

Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (Km ∼ 2 µm), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ∼1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg(2+) ion in a manner that allows it to assist in transition state stabilization. The Mg(2+) ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg(2+)·ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg(2+) coordination in the human cytosolic (Hc)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in HcTrpRS with a yet-to-be determined role for Mg(2+). Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to HcTrpRS.

Primary mucosal CD30-positive T-cell lymphoproliferative disorders of the head and neck rarely involving epiglottis: clinicopathological, immunohistomchemical and genetic features of a case.

A case of primary mucosal CD30-positive T-cell lymphoproliferative disorder of the head and neck rarely involving epiglottis in a 59-year-old male was reported. Histologically, the ulcerative mucosa was affected by sheets of mixed inflammatory infiltration, with scattered large atypical lymphoid cells arranging in an individual or small clusters with focal epidermotropism. Immunohistochemically, tumor cells were uniformly immunoreactive to antibodies against CD2, CD3, CD7, CD43, CD4, TIA-1, with a heterogeneous expression of CD30, but negative for CD20, CD79a, CD21, CD8, CD56, ALK, EMA, granzyme B. Epstein-Barr virus encoded RNA (EBER) were detected. Genetically, T-cell receptor (TCR) γ gene showed an oligoclonal rearrangement. This first case developing in epiglottis demonstrates mucosal CD30-positive T-cell lymphoproliferative disorders are characteristic of a broad clinicopathologic spectrum similar to the counterpart in the skin with a favorable prognosis.

Spindle cell/sclerosing rhabdomyosarcoma: case series from a single institution emphasizing morphology, immunohistochemistry and follow-up.

Spindle cell/sclerosing rhabdomyosarcoma is a rare skeletal-muscle tumor with distinctive clinicopathologic characteristics. 10 cases (6 cases of spindle cell rhabdomyosarcoma and 4 cases of scleroisng rhabdomyosarcoma) were composed of 6 males and 4 females aging from 5 months to 57 years, with median age 33 years, most of who represented a painless solid mass. Histologically, the tumors were composed of fascicles of spindle cells or primitive round cells embed in sclerotic matrix with presence of rhabdomyoblasts in varying proportion. Immunohistochemically, the tumor cells expressed MyoD1 (10/10), Desmin (10/10), myogenin (6/10), AE1/AE3 (2/10), EMA (2/10), but were negative for SMA, caldesmon, S-100. All of the patients underwent a complete surgical resection without or with chemotherapy (2/10) or radiotherapy (1/10). During the follow-up period (1 to 24 months), 1 patient was succumbed, and 2 cases showed in situ recurrence with 1 of them adopting metastasis. Our cases further demonstrate there do present some clincopathologic relations between spindle cells rhabdomyosarcoma and sclerosing rhabdomyosarcoma, but the latter seems to have a better prognosis. Exact grading and staging contribute to predict the outcome.

The effects of vascular endothelial growth factor C knockdown in esophageal squamous cell carcinoma.

PURPOSE: We investigated the role of vascular endothelial growth factor C (VEGF-C) in esophageal squamous cell carcinoma (ESCC) by knocking down VEGF-C expression in the ESCC cell line EC9706. METHODS: Immunohistochemistry and in situ hybridization techniques were used to detect the expression of VEGF-C expression in ESCC tissues. We also investigated the relationship between VEGF-C expression and lymph node metastasis. We designed a siRNA expression plasmid for VEGF-C and transfected it into EC9706 cells. Stable clones were selected, and VEGF-C expression was analyzed by RT-PCR and western blotting. Cells were inoculated into nude mice. The expression of VEGF-C in the resulting tumors was analyzed by immunohistochemistry and in situ hybridization. RESULTS: VEGF-C is highly expressed in ESCC and correlated with lymph node metastasis, as high levels were observed in patients presenting with lymph node metastases relative to those who did not (P < 0.01). Transfection with VEGF-C-siRNA decreased the expression of VEGF-C mRNA and protein. ESCC cells stably transfected with VEGF-C-siRNA expressed very low levels of VEGF-C (P < 0.01 compared with control). This knockdown effect persisted when the cells were inoculated into nude mice and allowed to form tumors. CONCLUSIONS: The siRNA-targeted knockdown of VEGF-C led to a significant reduction in VEGF-C expression. This siRNA technique could be used for gene therapy in ESCC.

CD44(+)/CD24(-) cells are transit progenitors and do not determine the molecular subtypes and clinical parameters in breast carcinomas.

CD44(+)/CD24(-) cells have been associated with breast cancer stem/progenitor cell features. However, the status of this phenotype cells in normal, benign and malignant breast tissues has not been studied, and the clinical correlation of this subpopulation in breast cancer is not fully understood. The present study sought to identify these cells in a series of normal, benign, and malignant breast tissues and explore their correlation to the molecular subtypes of breast carcinoma and conventional pathological features. Double-staining immunohistochemistry (DIHC) of CD44 and CD24 was performed on 30 normal breast tissues, 30 breast fibroadenomas (FA), 60 breast invasive ductal carcinomas (IDC), and 3 breast cancer cell lines (MCF-7, MDA-MB-468, and MDA-MB-231). In the normal breast tissues and FAs, three phenotypes were observed including CD44(+)/CD24(+), CD44(+)/CD24(-), and CD44(-)/CD24(-) cells. In the IDCs, CD44(-)/CD24(+) cells were detected, in addition to the three aforementioned phenotypes. The strong positive rate (+++, incidence >60%) of CD44(+)/CD24(-) was significantly increased from normal breast tissue, FAs to IDCs (0.0%-->6.7%-->21.7%). However, the CD44(+)/CD24(-) cells didn't correlate with ages of patients, lymph node metastasis, tumor size, molecular subtypes, and the expression of ER, PR, HER-2, PS2, Bcl-2, nm23. The proportion of CD44(+)/CD24(-) cells in MCF-7, MDA-MB-468, and MDA-MB-231 was about 1, 5, and 80%, respectively. The results indicate that the CD44(+)/CD24(-) cells are transit progenitors and have no association with the molecular subtypes and clinicopathological parameters in the IDCs.

Interconversion of ATP binding and conformational free energies by tryptophanyl-tRNA synthetase: structures of ATP bound to open and closed, pre-transition-state conformations.

Binding ATP to tryptophanyl-tRNA synthetase (TrpRS) in a catalytically competent configuration for amino acid activation destabilizes the enzyme structure prior to forming the transition state. This conclusion follows from monitoring the titration of TrpRS with ATP by small angle solution X-ray scattering, enzyme activity, and crystal structures. ATP induces a significantly smaller radius of gyration at pH=7 with a transition midpoint at approximately 8mM. A non-reciprocal dependence of Trp and ATP dissociation constants on concentrations of the second substrate show that Trp binding enhances affinity for ATP, while the affinity for Trp falls with the square of the [ATP] over the same concentration range ( approximately 5mM) that induces the more compact conformation. Two distinct TrpRS:ATP structures have been solved, a high-affinity complex grown with 1mM ATP and a low-affinity complex grown at 10mM ATP. The former is isomorphous with unliganded TrpRS and the Trp complex from monoclinic crystals. Reacting groups of the two individually-bound substrates are separated by 6.7A. Although it lacks tryptophan, the low-affinity complex has a closed conformation similar to that observed in the presence of both ATP and Trp analogs such as indolmycin, and resembles a complex previously postulated to form in the closely-related TyrRS upon induced-fit active-site assembly, just prior to catalysis. Titration of TrpRS with ATP therefore successively produces structurally distinct high- and low-affinity ATP-bound states. The higher quality X-ray data for the closed ATP complex (2.2A) provide new structural details likely related to catalysis, including an extension of the KMSKS loop that engages the second lysine and serine residues, K195 and S196, with the alpha and gamma-phosphates; interactions of the K111 side-chain with the gamma-phosphate; and a water molecule bridging the consensus sequence residue T15 to the beta-phosphate. Induced-fit therefore strengthens active-site interactions with ATP, substantially intensifying the interaction of the KMSKS loop with the leaving PP(i) group. Formation of this conformation in the absence of a Trp analog implies that ATP is a key allosteric effector for TrpRS. The paradoxical requirement for high [ATP] implies that Gibbs binding free energy is stored in an unfavorable protein conformation and can then be recovered for useful purposes, including catalysis in the case of TrpRS.