Reshaping Phosphatase Substrate Preference for Controlled Biosynthesis Using a "Design-Build-Test-Learn" Framework.

Biosynthesis is the application of enzymes in microbial cell factories and has emerged as a promising alternative to chemical synthesis. However, natural enzymes with limited catalytic performance often need to be engineered to meet specific needs through a time-consuming trial-and-error process. This study presents a quantum mechanics (QM)-incorporated design-build-test-learn (DBTL) framework to rationally design phosphatase BT4131, an enzyme with an ambiguous substrate spectrum involved in N-acetylglucosamine (GlcNAc) biosynthesis. First, mutant M1 (L129Q) is designed using force field-based methods, resulting in a 1.4-fold increase in substrate preference (kcat/Km) toward GlcNAc-6-phosphate (GlcNAc6P). QM calculations indicate that the shift in substrate preference is caused by a 13.59 kcal mol-1 reduction in activation energy. Furthermore, an iterative computer-aided design is conducted to stabilize the transition state. As a result, mutant M4 (I49Q/L129Q/G172L) with a 9.5-fold increase in kcat-GlcNAc6P/Km-GlcNAc6P and a 59% decrease in kcat-Glc6P/Km-Glc6P is highly desirable compared to the wild type in the GlcNAc-producing chassis. The GlcNAc titer increases to 217.3 g L-1 with a yield of 0.597 g (g glucose)-1 in a 50-L bioreactor, representing the highest reported level. Collectively, this DBTL framework provides an easy yet fascinating approach to the rational design of enzymes for industrially viable biocatalysts.

MicroRNA-1 promotes cartilage matrix synthesis and regulates chondrocyte differentiation via post-transcriptional suppression of Ihh expression.

Indian hedgehog signaling molecule (Ihh) is known to play critical roles in chondrogenesis and cartilage development. However, it remains largely unknown how Ihh is regulated during the process. Previous studies suggest that Ihh plays an important regulatory role in the growth and development of articular cartilage, but whether it is regulated by miRNAs is unclear. The present study aimed to investigate the effects of miR­1 on chondrocyte differentiation and matrix synthesis, and to determine whether miR­1 can regulate the Ihh signaling pathway. In the present study, the expression level of miR­1 was altered via transfection of the miR­1 mimic or inhibitor in mouse thorax chondrocytes, and the impact on chondrocyte phenotypes and Ihh expression was examined. Overexpression of miR­1 promoted the expression of the matrix synthesis­associated molecules collagen (Col)­II and aggrecan, two key components in cartilage matrix. Conversely, overexpression of miR­1 significantly downregulated the expression of chondrocyte differentiation markers Col­X and matrix metallopeptidase 13. Moreover, overexpression of miR­1 dose­dependently inhibited endogenous Ihh expression, and an association was observed between miR­1 and Ihh expression. The 3' untranslated region (UTR) of Ihh from various species contains two miR­1 binding sites. Luciferase reporter assays indicated that miR­1 post­transcriptionally suppressed Ihh expression, which was dependent on the binding of miR­1 to one of the two putative binding sites of the Ihh 3'UTR. Furthermore, via inhibition of Ihh expression, miR­1 decreased the expression of molecules downstream of Ihh in the Hedgehog signaling pathway in mouse thorax chondrocytes. This study provided new insight into the molecular mechanisms of miR­1 in regulating chondrocyte phenotypes via targeting the Ihh pathway.

Autografts vs Synthetics for Cruciate Ligament Reconstruction: A Systematic Review and Meta-Analysis.

To describe the outcomes of autografts and synthetics in anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) reconstruction with respect to instrumented laxity measurements, patient-reported outcome scores, complications, and graft failure risk. We searched PubMed, Cochrane Library, and EMBASE for published randomized controlled trials (RCT) and case controlled trials (CCTs) to compare the outcomes of the autografts versus synthetics after cruciate ligament reconstruction. Data analyses were performed using Cochrane Collaboration RevMan 5.0. Nine studies were identified from the literature review. Of these studies, three studies compared the results of bone-patellar tendon-bone (BPTB) and ligament augmentation and reconstruction system (LARS), while six studies compared the results of four-strand hamstring tendon graft (4SHG) and LARS. The comparative study showed no difference in Lysholm score and failure risk between autografts and synthetics. The combined results of the meta-analysis indicated that there was a significantly lower rate of side-to-side difference > 3 mm (Odds Ratio [OR] 2.46, 95% confidence intervals [CI] 1.44-4.22, P = 0.001), overall IKDC (OR 0.40, 95% CI 0.19-0.83, P = 0.01), complications (OR 2.54, 95% CI 1.26-5.14, P = 0.009), and Tegner score (OR -0.31, 95% CI -0.52-0.10, P = 0.004) in the synthetics group than in the autografts group. This systematic review comparing long-term outcomes after cruciate ligament reconstruction with either autograft or synthetics suggests no significant differences in failure risk. Autografts were inferior to synthetics with respect to restoring knee joint stability and patient-reported outcome scores, and were also associated with more postoperative complications.

Positive Effects of a Young Systemic Environment and High Growth Differentiation Factor 11 Levels on Chondrocyte Proliferation and Cartilage Matrix Synthesis in Old Mice.

OBJECTIVE: To investigate the effects of a young systemic environment and growth differentiation factor 11 (GDF-11) on aging cartilage. METHODS: A heterochronic parabiosis model (2-month-old mouse and 12-month-old mouse [Y/O]), an isochronic parabiosis model (12-month-old mouse and 12-month-old mouse [O/O]), and 12-month-old mice alone (O) were evaluated. Knee joints and chondrocytes from old mice were examined by radiography, histology, cell proliferation assays, immunohistochemistry, Western blotting, and quantitative reverse transcriptase-polymerase chain reaction 16 weeks after parabiosis surgery. GDF-11 was injected into 12-month-old mouse joints daily for 16 weeks. Cartilage degeneration, cell proliferation, and osteoarthritis-related gene expression were evaluated. RESULTS: Osteoarthritis Research Society International scores in old mice were significantly lower in the Y/O group than in the O/O and O groups (both P < 0.05). The percentage of 5-ethynyl-2'-deoxyuridine-positive chondrocytes in old mice was significantly higher in the Y/O group than in the other groups (P < 0.05). Type II collagen (CII) and SOX9 messenger RNA levels differed in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). RUNX-2, CX, and matrix metalloproteinase 13 levels were significantly lower in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). Similar results were obtained for protein expression levels and after GDF-11 treatment in vitro and in vivo. Phosphorylated Smad2/3 (pSmad2/3) levels were higher in the recombinant GDF-11-treated group than in the control group. CONCLUSION: A young systemic environment promotes chondrocyte proliferation and cartilage matrix synthesis in old mice. GDF-11, a "young factor," contributes to these effects through the up-regulation of pSmad2/3.

Adenovirus-mediated transduction with Histone Deacetylase 4 ameliorates disease progression in an osteoarthritis rat model.

BACKGROUND: Downregulation of histone deacetylase-4 (HDAC4) contributes to cartilage degeneration in osteoarthritis (OA) because it promotes upregulation of runt-related transcription factor-2 (Runx-2) and osteoarthritis-related genes. The effect of HDAC4 upregulation on cartilage damage in OA remains unknown. METHODS: Rat chondrocytes were infected with Ad-GFP or Ad-HDAC4-GFP for 48 h, stimulated with interleukin-1ß (IL-1ß, 10 ng/mL) for 24 h, and then harvested for RT-qPCR. Male Sprague-Dawley rats in 3 groups were given anterior cruciate ligament transection (ACLT) or sham operation, and knee injections with different adenovirus (Ad) vectors at 48 h after surgery and every 3 weeks thereafter: ACLT+Ad-GFP (n = 17); ACLT+Ad-HDAC4-GFP (n = 20); and sham+Ad-GFP (n = 15). Three ACLT-Ad-HDAC4-GFP rats were sacrificed at different times to examine the expression of HDAC4. Two ACLT-Ad-GFP rats and two ACLT-Ad-HDAC4-GFP rats were euthanized at week-2; articular cartilage was harvested and expression of HDAC4 was determined by RT-qPCR. All other rats were euthanized at week-8. Cartilage damage and OA progression was assessed using radiography, fluorescence molecular tomography (FMT), histology, immunohistochemistry (IHC), ELISA, and RT-qPCR. RESULTS: Overexpression of HDAC4 in chondrocytes stimulated by IL-1ß reduced the levels of Runx-2, MMP-13, and Collagen X, but increased the levels of Collagen II and Aggrecan. Upregulation of HDAC4 reduced osteophyte formation and cartilage damage, and increased articular cartilage anabolism. CONCLUSION: HDAC4 attenuated articular cartilage damage by repression of Runx-2, MMP-13, and collagen X and induction of collagen II and ACAN in this rat model of OA. Upregulation of HDAC4 may provide chondroprotection in OA patients.

Involvement of Aryl Hydrocarbon Receptor in L-Kynurenine-Mediated Parathyroid Hormone-Related Peptide Expression.

Parathyroid hormone-related peptide (PTHrP), produced by specific cancers such as lung cancer, profoundly influences the formation of bone metastatic lesions via the "vicious cycle" of tumor growth and bone resorption. The changes in gene expression regulated by the abnormal microenvironment components play key roles in maintaining the biological characteristics of cells, such as the organotropism of cancer metastasis. A recent study has shown that L-kynurenine (L-Kyn), one of microenvironment components, induced a substantial increase in the metastasis of lung cancer cells. What remains unclear, however, is the linkage between L-Kyn and bone metastatic lesions. In the present paper, we found that a significant upregulation of PTHrP expression was detected when 95D cells, a lung cancer cell line, were incubated with 50 µM of L-Kyn. Meanwhile, L-Kyn (50/100 µM) strongly strengthened aryl hydrocarbon receptor (Ahr) expression. Additionally, L-Kyn (50 µM) increased the expression of the nuclear translocation of Ahr and cytochrome P450 1A1. Most importantly, the L-Kyn-induced upregulation of migration was significantly reduced when cells were co-incubated with siRNAAhr. Notably, the L-Kyn-mediated increase in PTHrP was also substantially attenuated upon siRNAAhr treatment in 95D cells. These results suggest that Ahr is involved in the L-Kyn-induced enhancement of PTHrP expression.

Contribution of IL-1ß, 6 and TNF-α to the form of post-traumatic osteoarthritis induced by "idealized" anterior cruciate ligament reconstruction in a porcine model.

BACKGROUND: It has been noted that anterior cruciate ligament (ACL) injury-induced cartilage degeneration is the key risk factor for post-traumatic osteoarthritis (PTOA). However, whether the cartilage degeneration after ACL injury is caused by inflammation, abnormal biomechanics or both remains largely unknown, as there has been no animal model for separating the two factors so far. METHODS: Eighteen-month-old female mini-pigs were divided into an "idealized" anterior cruciate ligament reconstruction (IACLR) group and a control group (n = 16 limbs per group). Real-time PCR, safranine O staining and indian ink staining were performed to verify whether animal models were successfully established or not. Multiple linear regression analysis was used to evaluate the correlation between levels of the inflammatory factors (including interferon [IFN]-γ, interleukin [IL]-1ß, IL-4, IL-6, IL-8, IL-10, IL-12 and tumor necrosis factor [TNF]-α measured by the Luminex method) and changes in cartilage histology (quantified by morphological scoring) after surgery. RESULTS: A significant OA cartilage damage with increased MMP-1, MMP-13 mRNA levels and reduced aggrecan mRNA/protein levels was observed in IACLR groups. As a result, the IACLR gross morphology score was dramatically increased than control. Moreover, IACLR significantly increased the levels of IL-1ß, IL-4, IL-6 and TNF-α in the synovial fluid of the knee. Most importantly, a close relationship was found between IL-1ß, IL-6 and TNF-α concentrations and morphological score of PTOA, respectively. CONCLUSION: These results demonstrated that inflammatory factors are independently responsible for the onset of PTOA.