BIT: Bayesian Identification of Transcriptional Regulators.

BIT is a novel Bayesian hierarchical model capable of predicting transcriptional regulators (TRs) from the input of user-provided epigenomic regions. TRs are critical molecules in transcriptional regulation. Many diseases and cancers are linked to the dysfunction of TRs. Knowing TRs in certain biological process can help find new biomarkers or therapeutic targets. Thus, BIT formulates a novel Bayesian hierarchical model with the Pólya-gamma data augmentation strategy. Based on collected ChIP-seq datasets, BIT can identify TRs responsible for the genome-wide binding pattern within the user-provided epigenomic regions. BIT has been validated by using a simulation study and three applications.

FGF19-Based Mini Probe Targeting FGFR4 for Diagnosis and Surgical Navigation of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a frequent malignancy that has a high death rate and a high rate of recurrence following surgery, owing to insufficient surgical resection. Furthermore, HCC is prone to peritoneal metastasis (HCC-PM), resulting in a significant number of tiny cancer lesions, making surgical removal more challenging. As a potential imaging target, FGFR4 is highly expressed in tumors, especially in HCC, but is less expressed in the normal liver. In this study, we used computational simulation approaches to develop peptide I0 derived from FGF19, a particular ligand of FGFR4, and labeled it with the NIRF dye, MPA, for HCC detection. In surgical navigation, the TBR was 9.31 ± 1.36 and 8.57 ± 1.15 in HepG2 in situ tumor and HCC-PM models, respectively, indicating considerable tumor uptake. As a result, peptide I0 is an excellent clinical diagnostic reagent for HCC, as well as a tool for surgically resecting HCC peritoneal metastases.

Effect of edible oil type on the formation of protein-bound Nε-(carboxymethyl)lysine in roasted pork patties.

Protein-bound Nε-(carboxymethyl)lysine (CML), an advanced glycation end product within meat products, poses a potential health risk to humans. The objective of this study was to explore the impact of various edible oils on the formation of protein-bound CML in roasted pork patties. Eleven commercially edible oils including lard oil, corn oil, palm oil, olive oil, flaxseed oil, blended oil, camellia oil, walnut oil, soybean oil, peanut oil, and colza oil were added to pork tenderloin mince, respectively, at a proportion of 4 % to prepare raw pork patties. The protein-bound CML contents in the pork patties were determined by HPLC-MS/MS before and after roasting at 200 °C for 20 min. The results indicated that walnut oil, flaxseed oil, colza oil, olive oil, lard oil, corn oil, blended oil, and palm oil significantly reduced the accumulation of protein-bound CML in pork patties, of which the inhibition rate was in the 24.43 %-37.96 % range. Moreover, the addition of edible oil contributed to a marginal reduction in the loss of lysine. Meanwhile, glyoxal contents in pork patties were reduced by 16.72 %-43.21 % after roasting. Other than blend oil, all the other edible oils restrained protein oxidation in pork patties to varying degrees (between 20.16 % and 61.26 %). In addition, camellia oil, walnut oil, and flaxseed oil increased TBARS values of pork patties by 2.2-8.6 times when compared to the CON group. After analyzing the fatty acid compositions of eleven edible oils, five main fatty acids (palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid) were selected to establish Myofibrillar protein-Glucose-fatty acids systems to simulate the roasting process. The results showed that palmitic acid, oleic acid, linoleic acid, and linolenic acid obviously mitigated the formation of myofibrillar protein-bound CML, exhibiting suppression rates ranging from 10.38 % to 40.32 %. In conclusion, the addition of specific edible oil may curb protein-bound CML production in roasted pork patty by restraining protein or lipid oxidation, reducing lysine loss, and suppressing glyoxal production, which may be attributed to the fatty acid compositions of edible oils. This finding provides valuable guidance for the selection of healthy roasting oils in the thermal processing of meat products.

Acceptable Fusion Rate of Single-Level OLIF Using Pure Allograft Combined with Posterior Instrumentation through the Wiltse Approach: A 2-Year Follow-Up Study.

OBJECTIVE: Autogenic bone grafts have shown successful fusion rates in the treatment of degenerative lumbar disorders, but taking too many autogenic bones may result in donor site ischemia or infection. This study aimed to evaluate the outcomes of single-level oblique lumbar interbody fusion (OLIF) using pure allograft combined with posterior pedicle screw instrumentation through the Wiltse approach. METHODS: A retrospective case analysis was performed on a series of consecutive patients who received a single-level OLIF procedure combined with posterior pedicle screw instrumentation through the Wiltse approach between July 1, 2017, and December 31, 2019, in which pure allogenic bone graft was used and filled in the large window of the cage. The patients were followed up as scheduled at 1 day and 3, 6, 12, 24 months after operation. Clinical outcome was assessed by multiple questionnaires, including Oswestry disability index (ODI), Japanese Orthopaedic Association (JOA) score rating system, short form-36 health survey (SF-36), and visual analog scale (VAS) for low back pain. Radiographic outcome was evaluated by measuring the parameters such as disc height, lumbar lordosis, and segmental angle on the standard standing lateral radiographs, and the space angle of the fusion level on the dynamic views of the lateral radiographs. Subsidence of the cage and intervertebral fusion status were evaluated on both the radiographic and CT scan images. RESULTS: A total of 34 patients were finally included in this study. At 2-year follow-up, the VAS for low back pain, ODI, JOA, and SF-36 scores all had significant improvement (p < 0.001). Substantial increase of anterior and posterior disc heights was observed (p < 0.001). Both lumbar lordosis and segmental angle became larger (p < 0.05). No visible change of the space angle of the fusion level was found on the dynamic views. The 1-year fusion rate of 73.5% on CT scans proceeded to 82.4% at 2-year follow-up. The fusion rate was as high as 91.2% according to Bridwell interbody fusion grading system on radiographic images. The clinical outcomes in patients with incomplete fusion were just as good as those with complete fusion. The six patients with cage subsidence had higher ODI (p < 0.001) and lower JOA (p < 0.001) and SF-36 PCS (p = 0.011) scores than those without cage subsidence. CONCLUSION: The use of pure allograft in single-level OLIF resulted in an acceptable fusion rate and satisfactory clinical effect at 2-year follow-up. Supplementation of posterior pedicle screw through the minimally invasive Wiltse approach ensured the favorable outcomes both clinically and radiographically.

Smad7 Is Highly Expressed in Human Degenerative Discs and Participates in IL-1ß-Induced Apoptosis of Rat AF Cells via the Mitochondria Pathway.

Background: Abnormal Smad7 expression can lead to apoptosis in different cell types. Previously, we found high expression of Smad7 in rat degenerative discs. However, the exact role of Smad7 in the apoptosis of disc cells and the possible underlying mechanism remain unclear. Methods: Degenerative and nondegenerative human lumbar intervertebral discs were collected from patients during operation. The expressions of SMAD7 mRNA and protein in the different components of these discs were measured with real-time PCR and Western blotting, respectively. Annulus fibrosus (AF) cells were isolated and cultivated from the discs of young healthy rats. Smad7 in the AF cells was overexpressed with adenovirus and knocked down with siRNA. IL-1ß was used to induce apoptosis in the AF cells. Loss-and-gain cell function experiments were performed to show the effect of Smad7 on the apoptosis of AF cells. The function recovery experiments were performed to verify whether Smad7 regulates the apoptosis of AF cells through the mitochondria-mediated pathway. Results: Both the mRNA and protein expressions of Smad7 were significantly higher in the different components of human degenerative discs than in those of the nondegenerative discs. IL-1ß stimulated apoptosis while upregulating the Smad7 expression in the AF cells in vitro. Overexpression of Smad7 in AF cells exaggerated the IL-1ß-induced apoptosis in the cells while knockdown of Smad7 expression suppressed this apoptosis. With the exaggerated apoptosis in the AF cells with Smad7 overexpression, both active cleaved caspase-3 and cleaved caspase-9, the ratio of Bax/Bcl-2, and Cyt-c increased significantly. However, the inhibitor of caspase-9, Z-LEHD-FMK, significantly diminished the apoptosis in these cells. Conclusion: Smad7 is highly expressed in human degenerative discs and participates in IL-1ß-induced apoptosis of rat AF cells via the mitochondria pathway. Smad7 may be a potential target for the prevention and treatment of degenerative disc disease.

IgG-Engineered Protective Antigen for Cytosolic Delivery of Proteins into Cancer Cells.

Therapeutic immunotoxins composed of antibodies and bacterial toxins provide potent activity against malignant cells, but joining them with a defined covalent bond while maintaining the desired function is challenging. Here, we develop novel immunotoxins by dovetailing full-length immunoglobulin G (IgG) antibodies and nontoxic anthrax proteins, in which the C terminus of the IgG heavy chain is connected to the side chain of anthrax toxin protective antigen. This strategy enabled efficient conjugation of protective antigen variants to trastuzumab (Tmab) and cetuximab (Cmab) antibodies. The conjugates effectively perform intracellular delivery of edema factor and N terminus of lethal factor (LFN) fused with diphtheria toxin and Ras/Rap1-specific endopeptidase. Each conjugate shows high specificity for cells expressing human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR), respectively, and potent activity across six Tmab- and Cmab-resistant cell lines. The conjugates also exhibit increased pharmacokinetics and pronounced in vivo safety, which shows promise for further therapeutic development.

Targeting Cancer Gene Dependencies with Anthrax-Mediated Delivery of Peptide Nucleic Acids.

Antisense oligonucleotide therapies are important cancer treatments, which can suppress genes in cancer cells that are critical for cell survival. SF3B1 has recently emerged as a promising gene target that encodes a key splicing factor in the SF3B protein complex. Over 10% of cancers have lost one or more copies of the SF3B1 gene, rendering these cancers vulnerable after further suppression. SF3B1 is just one example of a CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) gene, but over 120 additional candidate CYCLOPS genes are known. Antisense oligonucleotide therapies for cancer offer the promise of effective suppression for CYCLOPS genes, but developing these treatments is difficult due to their limited permeability into cells and poor cytosolic stability. Here, we develop an effective approach to suppress CYCLOPS genes by delivering antisense peptide nucleic acids (PNAs) into the cytosol of cancer cells. We achieve efficient cytosolic PNA delivery with the two main nontoxic components of the anthrax toxin: protective antigen (PA) and the 263-residue N-terminal domain of lethal factor (LFN). Sortase-mediated ligation readily enables the conjugation of PNAs to the C terminus of the LFN protein. LFN and PA work together in concert to translocate PNAs into the cytosol of mammalian cells. Antisense SF3B1 PNAs delivered with the LFN/PA system suppress the SF3B1 gene and decrease cell viability, particularly of cancer cells with partial copy-number loss of SF3B1. Moreover, antisense SF3B1 PNAs delivered with a HER2-binding PA variant selectively target cancer cells that overexpress the HER2 cell receptor, demonstrating receptor-specific targeting of cancer cells. Taken together, our efforts illustrate how PA-mediated delivery of PNAs provides an effective and general approach for delivering antisense PNA therapeutics and for targeting gene dependencies in cancer.

[Study on the protective mechanism of autophagy on cartilage by magnesium sulfate].

Objective: To investigate the mechanism of magnesium sulfate in protecting rabbit cartilage by initiating autophagy. Methods: Twenty-four adult female New Zealand rabbits were used to prepare post-traumatic osteoarthritis (PTOA) models by anterior cruciate ligament transection. Then, the PTOA models were randomly divided into PTOA group, distilled water group, and magnesium sulfate group, with 8 rabbits in each group. Immediately after operation, the distilled water group and the magnesium sulfate group were injected with 0.5 mL distilled water and 20 mmol/L magnesium sulfate solution in the joint cavity 3 times a week for 4 weeks, respectively. The PTOA group was not treated. The general condition of the animals was observed after operation. After 4 weeks, the expressions of tumor necrosis factor α (TNF-α) and collagen typeⅡ in the joint fluid and the expression of collagen type Ⅱ in venous blood were detected by ELISA assay. The protein expressions of transient receptor potential channel vanilloid 5 (TRPV5) and microtubule associated protein 1 light chain 3 (LC3; LC3-Ⅱ/LC3-Ⅰ) in femoral cartilage were detected by Western blot. The mRNA expressions of interleukin 1ß (IL-1ß), TNF-α, matrix metalloproteinases 3 (MMP-3) in synovial tissue and collagen type Ⅱ, Aggrecan (AGN), SOX9 in cartilage tissue were detected by real-time fluorescence quantitative PCR. Cartilage tissue sections were stained with HE staining, Masson staining, and Alcian blue staining and scored according to the modified histological osteoarthritis (OA) score. Results: All animals survived until the experiment was completed. Compared with the other two groups, the expression of TNF-α in joint effusion and collagen type Ⅱ in joint effusion and venous blood were decreased in magnesium sulfate group; the protein expression of TRPV5 decreased, and the ratio of LC3-Ⅱ/LC3-Ⅰ increased significantly; the mRNA expressions of IL-1ß, TNF-α, and MMP-3 in synovial tissue were decreased, and the mRNA expressions of collagen type Ⅱ, AGN, and SOX9 in cartilage tissue were increased; OA scores also decreased significantly. All differences were statistically significant ( P<0.05). There was no significant difference in the above indicators between the PTOA group and the distilled water group ( P>0.05). Conclusion: Intra-articular injection of magnesium sulfate can reduce intra-articular inflammation, reduce the loss of collagen type Ⅱ and AGN, and is beneficial to cartilage regeneration in rabbits. The mechanism may be related to the initiation of chondroautophagy by inhibiting the calcium channel TRPV5.

Low-Molecular Weight Polyethylenimine Modified with Pluronic 123 and RGD- or Chimeric RGD-NLS Peptide: Characteristics and Transfection Efficacy of Their Complexes with Plasmid DNA.

To solve the problem of transfection efficiency vs. cytotoxicity and tumor-targeting ability when polyethylenimine (PEI) was used as a nonviral gene delivery vector, new degradable PEI polymers were synthesized via cross-linking low-molecular-weight PEI with Pluronic P123 and then further coupled with a targeting peptide R4 (RGD) and a bifunctional R11 (RGD-NLS), which were termed as P123-PEI-R4 and P123-PEI-R11, respectively. Agarose gel electrophoresis showed that both P123-PEI-R4 and P123-PEI-R11 efficaciously condense plasmid DNA at a polymer-to-pDNA w/w ratio of 3.0 and 0.4, respectively. The polyplexes were stable in the presence of serum and could protect plasmid DNA against DNaseI. They had uniform spherical nanoparticles with appropriate sizes around 100-280 nm and zeta-potentials about +40 mV. Furthermore, in vitro experiments showed that these polyplexes had lower cytotoxicity at any concentration compared with PEI 25 kDa, thus giving promise to high transfection efficiency as compared with another P123-PEI derivate conjugated with trifunctional peptide RGD-TAT-NLS (P123-PEI-R18). More importantly, compared with the other polymers, P123-PEI-R11 showed the highest transfection efficiency with relatively lower cytotoxicity at any concentration, indicating that the new synthetic polymer P123-PEI-R11 could be used as a safe and efficient gene deliver vector.

Cloning, expression, purification and characterization of an iron-dependent regulator protein from Thermobifida fusca.

Iron-dependent regulators (IdeRs) control the transcription of a variety of genes associated with iron homeostasis in Gram-positive bacteria. In this study we report the cloning of a putative IdeR gene from the moderate thermophile Thermobifida fusca into the pET-21a(+) expression vector. The expressed protein, Tf-IdeR, was purified using immobilized metal affinity and size-exclusion chromatography, and yielded approximately 12-16 mg of protein per liter of culture. The purified Tf-IdeR protein binds the tox operator sequence in the presence of divalent metal ions. Two Tf-IdeR binding sites were identified in the T. fusca genome upstream of a putative enterobactin exporter and a putative ABC-type multidrug transporter.