Dedifferentiation-like reprogramming of degenerative nucleus pulposus cells into notochordal-like cells by defined factors.

The extensive degeneration of functional somatic cells and the depletion of endogenous stem/progenitor populations present significant challenges to tissue regeneration in degenerative diseases. Currently, a cellular reprogramming approach enabling directly generating corresponding progenitor populations from degenerative somatic cells remains elusive. The present study focused on intervertebral disc degeneration (IVDD) and identified a three-factor combination (OCT4, FOXA2, TBXT (OFT)) that could induce the dedifferentiation-like reprogramming of degenerative nucleus pulposus cells (dNPCs) toward induced notochordal-like cells (iNCs). Single-cell transcriptomics dissected the transitions of cell identity during reprogramming. Further, OCT4 was found to directly interact with bromodomain PHD-finger transcription factor (BPTF) to remodel the chromatin during the early phases, which was crucial for initiating this dedifferentiation-like reprogramming. In rat models, intradiscal injection of adeno-associated virus carrying OFT generated iNCs from in situ dNPCs and reversed IVDD. These results collectively present a proof-of-concept for dedifferentiation-like reprogramming of degenerated somatic cells into corresponding progenitors through the development of a factor-based strategy, providing a promising approach for regeneration in degenerative disc diseases.

CSINet: A Cross-Scale Interaction Network for Lightweight Image Super-Resolution.

In recent years, advancements in deep Convolutional Neural Networks (CNNs) have brought about a paradigm shift in the realm of image super-resolution (SR). While augmenting the depth and breadth of CNNs can indeed enhance network performance, it often comes at the expense of heightened computational demands and greater memory usage, which can restrict practical deployment. To mitigate this challenge, we have incorporated a technique called factorized convolution and introduced the efficient Cross-Scale Interaction Block (CSIB). CSIB employs a dual-branch structure, with one branch extracting local features and the other capturing global features. Interaction operations take place in the middle of this dual-branch structure, facilitating the integration of cross-scale contextual information. To further refine the aggregated contextual information, we designed an Efficient Large Kernel Attention (ELKA) using large convolutional kernels and a gating mechanism. By stacking CSIBs, we have created a lightweight cross-scale interaction network for image super-resolution named "CSINet". This innovative approach significantly reduces computational costs while maintaining performance, providing an efficient solution for practical applications. The experimental results convincingly demonstrate that our CSINet surpasses the majority of the state-of-the-art lightweight super-resolution techniques used on widely recognized benchmark datasets. Moreover, our smaller model, CSINet-S, shows an excellent performance record on lightweight super-resolution benchmarks with extremely low parameters and Multi-Adds (e.g., 33.82 dB@Set14 × 2 with only 248 K parameters).

Design, synthesis, and biological evaluation of a potent PLK4 inhibitor WY29 with 1H-pyrazolo[3,4-d]pyrimidine scaffold.

Centriole duplication occurs once per cell cycle and is regulated by Polo-like kinase 4 (PLK4). Overexpression of PLK4 in somatic cells can lead to the excessive formation of centrioles, directly causing chromosome segregation errors and tumorigenesis. In this study, we described our efforts to develop a series of PLK4 inhibitors with 1H-pyrazolo[3,4-d]pyrimidine core, and further structure- and receptor-based design and optimization resulted in a potent inhibitor WY29 (IC50 = 0.027 µM), which exhibited good selectivity to other PLK family members (PLK1-3). At the cellular level, compound WY29 showed excellent antiproliferative activity against three breast cancer cell lines (MCF-7, BT474, and MDA-MB-231) while weak inhibitory activity was found on normal cell line HUVECs. In addition, the in vitro preliminary drug-like properties evaluation of compound WY29 showed outstanding stability in human plasma and liver microsomes, and weak inhibitory activity against the major subtypes of human cytochrome P450. Also, the drug-like properties prediction of compound WY29 displayed remarkable drug-like properties (drug-likeness mode score: 1.06). In conclusion, these results support the further development of compound WY29 as a lead compound for PLK4-targeted anticancer drug discovery.

[Efficiency and safety of microsurgical cluster ligation of the spermatic vein].

OBJECTIVE: To study the effect and safety of microscopic varicocele cluster ligation (MVCL). METHODS: We selected 28 patients undergoing bilateral microscopic spermatic vein ligation in Xuzhou Central Hospital from July 2021 to June 2022. Using the computerized randomization method, we performed MVCL or microscopic varicocele ligation (MVL) for the right or the left spermatic cord, respectively. We recorded the operation time, intraoperative blood loss, the numbers of the spermatic veins ligated and the arteries and lymphatic vessels preserved in each surgical side. A surgeon unaware of the surgical approach on the operative side collected the Visual Analogue Scale (VAS) pain scores, nodular foreign body sensation, relief of scrotal cramps, complications, and long-term recurrence from the patients. RESULTS: Compared with the MVL group, the MVCL group showed significantly shorter time for spermatic vein ligation (ï¼»56.21±13.96ï¼½ vs ï¼»31.43±10.13ï¼½ min, P<0.01), lower VAS scores on the 1st postoperative day (P <0.05) and a lower incidence of intra-scrotal nodular foreign body sensation in the 1st postoperative month (P <0.05). There were no statistically significant differences in the intraoperative blood loss, numbers of spermatic veins ligated and arteries and lymphatic vessels preserved, VAS scores at 3 and 7 postoperative days, incidence of complications and long-term recurrence between the two groups (P >0.05). CONCLUSION: MVCL is superior to MVL in reducing the time of spermatic vein ligation and improving the efficiency, efficacy and safety of the procedure, and therefore worthy of clinical promotion.

Development of a Fast Raman-Assisted Antibiotic Susceptibility Test (FRAST) for the Antibiotic Resistance Analysis of Clinical Urine and Blood Samples.

Human health is at great risk due to the spreading of antimicrobial resistance (AMR). The lengthy procedure of conventional antimicrobial susceptibility testing (AST) usually requires a few days. We developed a fast Raman-assisted antibiotic susceptibility test (FRAST), which detects single bacterial metabolic activity in the presence of antibiotics, using Raman single-cell spectroscopy. It was found that single-cell Raman spectra (SCRS) would show a clear and distinguishable Raman band at the "silent zone" (2000-2300 cm-1), due to the active incorporation of deuterium from heavy water (D2O) by antibiotic-resistant bacteria. This pilot study has compared the FRAST and the conventional AST for six clinical standard quality controls (four Gram-negative and two Gram-positive bacteria strains) in response to 38 antibiotics. In total, 3200 treatments have been carried out and approximately 64 000 SCRS have been acquired for FRAST analysis. The result showed an overall agreement of 88.0% between the FRAST and the conventional AST assay. The gram-staining classification based on the linear discriminant analysis (LDA) model of SCRS was developed, seamlessly coupling with the FRAST to further reduce the turnaround time. We applied the FRAST to real clinical analysis for nine urinary infectious samples and three sepsis samples. The results were consistent with MALDI-TOF identification and the conventional AST. Under the optimal conditions, the "sample to report" of the FRAST could be reduced to 3 h for urine samples and 21 h for sepsis samples. The FRAST provides fast and reliable susceptibility tests, which could speed up microbiological analysis for clinical practice and facilitate antibiotic stewardship.

Scar Tissue-Targeting Polymer Micelle for Spinal Cord Injury Treatment.

Spinal cord injury (SCI) is a devastating disorder, leading to permanent motor and sensory deficit. Despite recent advances in neurosciences, the treatment efficacy on SCI patients remains unsatisfactory, mainly due to the poor accumulation, short retention, and lack of controlled release of therapeutics in lesion tissue. Herein, an injured spinal cord targeting prodrug polymer micelle is built. An esterase-responsive bond is used to link apocynin (APO) monomer, because of the enhanced esterase activity found in microglia cells after activation, which ensures a controlled degradation of APO prodrug (Allyloxypolyethyleneglycol-b-poly [2-(((4-acetyl-2-methoxyphenoxy)carbonyl)oxy)ethyl methacrylate], APEG-PAPO or PAPO) by activated microglia cells. A scar tissue-homing peptide (cysteine-alanine-glutamine-lysine, CAQK) is introduced to the PAPO to endow the polymer micelle the lesion tissue-targeting ability. As a result, this CAQK-modified prodrug micelle (cPAM) exhibits an improved accumulation and prolonged retention in lesion tissue compared to the control micelle. The cPAM also leads to superior tissue protection and sustained motor function recovery than the control groups in a mouse model of SCI. In conclusion, the cPAM induces an effective treatment of SCI by the lesion tissue specific delivery of the prodrug polymer via its robust scar binding effect, making the scar tissue a drug releasing platform for sustained treatment of SCI.

An injectable recombinant human milk fat globule-epidermal growth factor 8-loaded copolymer system for spinal cord injury reduces inflammation through NF-κB and neuronal cell death.

Spinal cord injury (SCI) is a common disease and a major cause of paralysis, carrying much burden around the world. Despite the progress made with growth factors therapy, the response rate of acute SCI treatment still remains unsatisfactory, due largely to complex and severe inflammatory reactions. Herein, we prepare a MFG-E8-loaded copolymer system-based anti-inflammation therapy for SCI treatment. It is shown that the MFG-E8-loaded copolymer system can decrease pro-inflammatory cytokine expression and neuron death. In a rat model of crush-caused SCI, the copolymer system shows significant therapeutic efficacy by ameliorating inflammation, decreasing fibrotic scar, promoting myelin regeneration and suppressing overall SCI severity.

Osmolarity controls the differentiation of adipose-derived stem cells into nucleus pulposus cells via histone demethylase KDM4B.

Adipose-derived stem cells (ADSCs) are an ideal source of cells for intervertebral disc (IVD) regeneration, but the effect of an increased osmotic microenvironment on ADSC differentiation remains unclear. Here, we aimed to elucidate whether hyperosmolarity facilitates ADSC nucleus pulposus (NP)-like differentiation and whether histone demethylase KDM4B is involved in this process. ADSCs were cultured under standard and increased osmolarity conditions for 1-3 weeks, followed by analysis for proliferation and viability. Differentiation was then quantified by gene and protein analysis. Finally, KDM4B knockdown ADSCs were generated using lentiviral vectors. The results showed that increasing the osmolarity of the differentiation medium to 400 mOsm significantly increased NP-like gene expression and the synthesis of extracellular matrix (ECM) components during ADSC differentiation; however, further increasing the osmolarity to 500 mOsm suppressed the NP-like differentiation of ADSCs. KDM4B, as well as the IVD formation regulators forkhead box (Fox)a1/2 and sonic hedgehog (Shh), were found to be significantly upregulated at 400 mOsm. KDM4B knockdown reduced Foxa1/2, Shh, and NP-associated markers' expression, as well as the synthesis of ECM components. The reduction in NP-like differentiation caused by KDM4B knockdown was partially rescued by Purmorphamine, a specific agonist of Shh. Moreover, we found that KDM4B can directly bind to the promoter region of Foxa1/2 and decrease the content of H3K9me3/2. In conclusion, our results indicate that a potential optimal osmolarity window might exist for successful ADSC differentiation. KDM4B plays an essential role in regulating the osmolarity-induced NP-like differentiation of ADSCs by interacting with Foxa1/2-Shh signaling.

FoxA2 regulates the type II collagen-induced nucleus pulposus-like differentiation of adipose-derived stem cells by activation of the Shh signaling pathway.

Adipose tissue-derived stem cell (ADSC)-based therapy is promising for the treatment of intervertebral disc (IVD) degeneration, but the difficulty in inducing nucleus pulposus (NP)-like differentiation limits its clinical applications. Forkhead box (Fox)-A2 is an essential transcription factor for the formation of a normal NP. We demonstrated that type II collagen stimulates NP-like differentiation of ADSCs, partly by increasing the expression of FoxA2. We constructed FoxA2-overexpressing and -knockdown ADSCs by using lentiviral vectors. FoxA2 overexpression significantly enhanced NP-specific gene expression and the synthesis of glycosaminoglycan and collagen, whereas FoxA2 knockdown decreased NP-like differentiation and the expression of aggrecan and collagen II. The enhanced NP-like differentiation related to FoxA2 overexpression was partially rescued by an Shh signaling pathway inhibitor. In addition, FoxA2 inhibited the expression of Itg-α2 and further promoted NP-like differentiation induced by type II collagen. Furthermore, FoxA2-overexpressing ADSCs combined with type II collagen hydrogels promoted regeneration of degenerated NP in vivo. Our findings suggest that FoxA2 plays an essential role in the NP-like differentiation of ADSCs by activating the Shh signaling pathway.-Zhou, X., Ma, C., Hu, B., Tao, Y., Wang, J., Huang, X., Zhao, T., Han, B., Li, H., Liang, C., Chen, Q., Li, F. FoxA2 regulates the type II collagen-induced nucleus pulposus-like differentiation of adipose-derived stem cells by activation of the Shh signaling pathway.

Investigation of Bonding Behavior of FRP and Steel Bars in Self-Compacting Concrete Structures Using Acoustic Emission Method.

To extend understanding of the bonding behavior of fiber reinforced polymer (FRP) and steel bars in self-compacting concrete (SCC), an experimental series consisting of 36 direct pull-out tests monitored by acoustic emission (AE) were performed in this paper. The test variables involved rebar type, bar diameter, embedded length, and polypropylene (PP) fiber volume content. For each test, the pull-out force and free end slip were continuously measured and compared with the corresponding AE signals. It was found that the proposed AE method was effective in detecting the debonding process between the FRP/steel bars and the hosting concrete. The AE signal strength exhibited a good correlation with the actual bond stress-slip relationship measured in each specimen. Based on the AE location technique, the invisible non-uniform distribution of bonding stress along the bar was further revealed, the initial location of damage and the debonding process were captured. Additionally, the contribution of bar-to-concrete load-bearing mechanism (chemical adhesion, friction, and mechanical interlocking) to sustain the pull-out force was effectively clarified by studying the collected signals in the frequency domain of AE methods. The experimental results demonstrate that the proposed AE method has potential to detect the debonding damage of FRP/steel bar reinforced SCC structures accurately.

The Influence of Hyperosmolarity in the Intervertebral Disc on the Proliferation and Chondrogenic Differentiation of Nucleus Pulposus-Derived Mesenchymal Stem Cells.

Nucleus pulposus-derived mesenchymal stem cells (NP-MSCs) are suitable cell candidates for intervertebral disc (IVD) regeneration. However, little work has been done to determine the proliferation and chondrogenic differentiation of NP-MSCs in the hyperosmotic microenvironment of IVD. This study aimed to investigate the influence of the hyperosmolarity of IVD on the proliferation and chondrogenic differ-entiation of NP-MSCs. NP-MSCs were cultured in media of 300, 400, 430, and 500 mOsm/L, mimicking the osmotic pressures of serious degenerative, moderately degenerative, and healthy IVD. Cell proliferation was measured by CCK-8 assay. The expression of aggrecan, collagen I, and collagen II were measured by gene and protein expression analysis. Alcian blue and dimethylmethylene blue assay were used to investigate the accumulation of sulfate glycosaminoglycan. The regulation role of extracellular signal-regulated kinase (ERK) pathway was also analyzed. The results showed that, compared to 300 mOsm/L, hyperosmolarity of healthy IVD (430 and 500 mOsm/L) inhibited the proliferation and chondrogenic differentiation of NP-MSCs. The relative hypoosmotic condition of moderately degenerative IVD (400 mOsm/L) led to great proliferation and chondrogenic differentiation capacity. The ERK pathway was activated by the hyperosmolarity; inhibition of the ERK pathway abolished the difference in cell proliferation between the 300 mOsm/L and the hyperosmotic conditions, and enhanced chondrogenic differentiation. In conclusion, hyperosmolarity of IVD had a significant impact on the proliferation and chondrogenic differentiation of NP-MSCs. The ERK pathway was involved in the inhibition of proliferation and chondrogenic differentiation of NP-MSCs by the hyperosmolarity of IVD. The relative hypo-osmotic condition prevailing in degenerative discs offers a more permissive microenvironment for NP-MSCs.

Wear Degree Quantification of Pin Connections Using Parameter-Based Analyses of Acoustic Emissions.

Pin connections are commonly used in many engineering fields, and continuous operation may cause severe wear on the pins and may lead to their eventual fracture, if undetected. However, a reliable nonintrusive real-time method to monitor the wear of pin connections is yet to be developed. In this paper, acoustic emission (AE)-based parametric analysis methods, including the logarithm of the cumulative energy (LAE), the logarithm of the slope of cumulative energy (LSCE), the b-value method, the Ib-value method, and the fast Fourier transformation (FFT), were developed to quantify the wear degree of pin connections. The b-value method offers a criterion to quickly judge whether severe wear occurs on a pin connection. To assist the research, an experimental apparatus to accelerate wear test of pin connections was designed and fabricated. The AE sensor, mounted on the test apparatus in a nondestructive manner, is capable of real-time monitoring. The micrographs of the wear of pins, and the surface roughness of pins, verified that the values of the max LAE and the max LSCE became larger as the wear degree of pin connections increased, which means different values of the max LAE and the max LSCE can reflect different wear degree of pin connections. Meanwhile, the results of the micrographs and surface roughness confirmed that the b-value is an effective method to identify severe wear, and the value "1" can be used as a criterion to detect severe damage in different structures. Furthermore, the results of spectrum analysis in the low frequency range showed that the wear frequency was concentrated in the range of 0.01 to 0.02 MHz for the pin connection. This study demonstrated that these methods, developed based on acoustic emission technique, can be utilized in quantifying the wear degree of pin connections in a nondestructive way.

Protective Effect of Quercetin against Oxidative Stress-Induced Cytotoxicity in Rat Pheochromocytoma (PC-12) Cells.

Oxidative stress has been implicated in the pathogenesis of many kinds of neurodegenerative disorders, particularly Parkinson's disease. Quercetin is a bioflavonoid found ubiquitously in fruits and vegetables, and has antioxidative activity. However, the underlying mechanism of the antioxidative effect of quercetin in neurodegenerative diseases has not been well explored. Here, we investigated the antioxidative effect and underlying molecular mechanisms of quercetin on PC-12 cells. We found that PC-12 cells pretreated with quercetin exhibited an increased cell viability and reduced lactate dehydrogenase (LDH) release when exposed to hydrogen peroxide (H2O2). The significantly-alleviated intracellular reactive oxygen species (ROS), malondialdehyde (MDA), and lipoperoxidation of the cell membrane of PC-12 cells induced by H2O2 were observed in the quercetin pretreated group. Furthermore, quercetin pretreatment markedly reduced the apoptosis of PC-12 cells and hippocampal neurons. The inductions of antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in PC-12 cells exposed to H2O2 were significantly reduced by preatment with quercetin. In addition, quercetin pretreatment significantly increased Bcl-2 expression, and reduced Bax, cleaved caspase-3 and p53 expressions. In conclusion, this study demonstrated that quercetin exhibited a protective effect against oxidative stress-induced apoptosis in PC-12 cells. Our findings suggested that quercetin may be developed as a novel therapeutic agent for neurodegenerative diseases induced by oxidative stress.

Changes in CO2 concentration drive a succession of toxic and non-toxic strains of Microcystis blooms.

The dynamic changes between toxic and non-toxic strains of Microcystis blooms have always been a hot topic. Previous studies have found that low CO2 favors toxic strains, but how changing dissolved CO2 (CO2 [aq]) in water body influences the succession of toxic and non-toxic strains in Microcystis blooms remains uncertain. Here, we combined laboratory competition experiments, field observations, and a machine learning model to reveal the links between CO2 changes and the succession. Laboratory experiments showed that under low CO2 conditions (100-150 ppm), the toxic strains could make better use of CO2 (aq) and be dominant. The non-toxic strains demonstrated a growth advantage as CO2 concentration increased (400-1000 ppm). Field observations from June to November in Lake Taihu showed that the percentage of toxic strains increased as CO2 (aq) decreased. Machine learning highlighted links between the inorganic carbon concentration and the proportion of advantageous strains. Our findings provide new insights for cyanoHABs prediction and prevention.

Design, Modeling, and Experimental Validation of an Active Microcatheter Driven by Shape Memory Effects.

Microcatheters capable of active guidance have been proven to be effective and efficient solutions to interventional surgeries for cardiovascular and cerebrovascular diseases. Herein, a novel microcatheter made of two biocompatible materials, shape memory alloy (SMA) and polyethylene (PE), is proposed. It consists of a reconfigurable distal actuator and a separate polyethylene catheter. The distal actuator is created via embedding U-shape SMA wires into the PE base, and its reconfigurability is mainly dominated by the shape memory effect (SME) of SMA wires, as well as the effect of thermal mismatch between the SMA and PE base. A mathematical model was established to predict the distal actuator's deformation, and the analytical solutions show great agreement with the finite element results. Structural optimization of such microcatheters was carried out using the verified analytical model, followed by fabrication of some typical prototypes. Experimental testing of their mechanical behaviors demonstrates the feasibility of the structural designs, and the reliability and accuracy of the mathematical model. The active microcatheter, together with the prediction model, will lay a solid foundation for rapid development and optimization of active navigation strategies for vascular interventions.

Research Advances in Stem Cell Therapy for Erectile Dysfunction.

Erectile dysfunction (ED) is a common clinical condition that mainly affects men aged over 40 years. Various causes contribute to the progression of ED, including pelvic nerve injury, diabetes, metabolic syndrome, age, Peyronie's disease, smoking, and psychological disorders. Current treatments for ED are limited to symptom relief and do not address the root cause. Stem cells, with their powerful ability to proliferate and differentiate, are a promising approach for the treatment of male ED and are gradually gaining widespread attention. Current uses for treating ED have been studied primarily in experimental animals, with most studies observing improvements in erectile quality as well as improvements in erectile tissue. However, research on stem cell therapy for human ED is still limited. This article summarizes the recent literature on basic stem cell research on ED, including cavernous nerve injury, aging, diabetes, and sclerosing penile disease, and describes mechanisms of action and therapeutic effects of various stem cell therapies in experimental animals. Stem cells are also believed to interact with host tissue in a paracrine manner, and improved function can be supported through both implantation and paracrine factors. To date, stem cells have shown some preliminary promising results in animal and human models of ED.

Micro-flow cell washing technique combined with single-cell Raman spectroscopy for rapid and automatic antimicrobial susceptibility test of pathogen in urine.

Facing the rapid spread of antimicrobial resistance, methods based on single-cell Raman spectroscopy have proven their advances in reducing the turn-around time (TAT) of antimicrobial susceptibility tests (AST). However, the Raman-based methods are still hindered by the prolonged centrifugal cell washing procedure, which may require complex labor operation and induce high mechanical stress, resulting in a pretreatment time of over 1 h as well as a high cell-loss probability. In this study, we developed a micro-flow cell washing device and corresponding Raman-compatible washing chips, which were able to automatically remove the impurities in the samples, retain the bacterial cell and perform Raman spectra acquisition in situ. Results of washing the 5- and 10-µm polymethyl methacrylate (PMMA) microspheres showed that the novel technique achieved a successful removal of 99 % impurity and an 80 % particle retention rate after 6 to 10 cycles of washing. The micro-flow cell washing technique could complete the pretreatment for urine samples in a 96-well plate within 10 min, only taking 15 % of the handling time required by centrifugation. The AST profiles of urine sample spiked with E. coli 25922, E. faecalis 29212, and S. aureus 29213 obtained by the proposed Raman-based approach were found to be 100 % consistent with the results from broth micro-dilution while reducing the TAT to 3 h from several days which is required by the latter. Our study has demonstrated the micro-flow cell washing technique is a reliable, fast and compatible approach to replace centrifuge washing for sample pretreatment of Raman-AST and could be readily applied in clinical scenarios.

Teth137, a Conserved Factor of Unknown Function from Thermoanaerobacter ethanolicus JW200, Represses the Transcription of the adhE Gene In Vitro.

Teth137, a 13.7 kD protein of unknown function from Thermoanaerobacter ethanolicus JW200, is encoded by 360 nucleotides and has been obtained by DNA-coupled column previously. However, no function study of Teth137 has been published. Homologous modeling of Teth137 shows the protein is comprised of a helix-turn-helix motif which is a typical DNA-binding domain. Therefore, it is speculated Teth137 is a DNA-binding protein and involved in transcription of the adhE gene (encodes alcohol dehydrogenase E). To investigate the function of Teth137, recombinant Teth137 is overexpressed in Escherichia coli JM109 and purified by DEAE column. Purified Teth137 exhibits the affinity with the adhE promoter region in gel electrophoresis mobility shift assay (GEMSA). Teth137 at the concentration of 48 µM retards the migration of 5 nM of probe in the presence of the competitor DNA. Mutant analysis indicates that S69, T70, P71 and T72 are critical to protein-DNA interface; Gly substitutions at these residues results in the loss of the binding ability with the adhE promoter region. Moreover, T. ethanolicus JW200 RNA polymerase, σ subunit and template plasmid are prepared for in vitro transcription assay to detect the regulation function of Teth137. The results of the in vitro transcription show that the transcription of 5 nM of the template plasmid is inhibited by 48 µM of Teth137.

Cultureless enumeration of live bacteria in urinary tract infection by single-cell Raman spectroscopy.

Urinary tract infections (UTIs) are the most common outpatient infections. Obtaining the concentration of live pathogens in the sample is crucial for the treatment. Still, the enumeration depends on urine culture and plate counting, which requires days of turn-around time (TAT). Single-cell Raman spectra combined with deuterium isotope probing (Raman-DIP) has been proven to identify the metabolic-active bacteria with high accuracy but is not able to reveal the number of live pathogens due to bacteria replication during the Raman-DIP process. In this study, we established a new approach of using sodium acetate to inhibit the replication of the pathogen and applying Raman-DIP to identify the active single cells. By combining microscopic image stitching and recognition, we could further improve the efficiency of the new method. Validation of the new method on nine artificial urine samples indicated that the exact number of live pathogens obtained with Raman-DIP is consistent with plate-counting while shortening the TAT from 18 h to within 3 h, and the potential of applying Raman-DIP for pathogen enumeration in clinics is promising.

Nucleus pulposus cell-derived efficient microcarrier for intervertebral disc tissue engineering.

Adipose-derived stem cells (ADSCs) show great potential for the treatment of intervertebral disc (IVD) degeneration. An ideal carrier is necessary to transplant ADSCs into degenerated IVDs without influencing cell function. Nucleus pulposus cells (NPCs) can synthesize and deposit chondroitin sulfate and type II collagen which are NP-specific extracellular matrix (ECM) and can also regulate the NP-specific differentiation of stem cells. Bioscaffolds fabricated based on the ECM synthesis functions of NPCs have possible roles in cell transplantation and differentiation induction, but it has not been studied. In this study, we first aggregated NPCs into pellets, and then, NPC-derived efficient microcarriers (NPCMs) were fabricated by pellet cultivation under specific conditions and optimized decellularization. Thirdly, we evaluated the microstructure, biochemical composition, biostability and cytotoxicity of the NPCMs. Finally, we investigated the NP-specific differentiation of ADSCs induced by the NPCMsin vitroand NP regeneration induced by the ADSC-loaded NPCMs in a rabbit model. The results indicated that the injectable NPCMs retained maximal ECM and minimal cell nucleic acid after optimized decellularization and had good biostability and no cytotoxicity. The NPCMs also promoted the NP-specific differentiation of ADSCsin vitro. In addition, the results of MRI, x-ray, and the structure and ECM content of NP showed that the ADSCs-loaded NPCMs can partly restored the degenerated NPin vivo. Our injectable NPCMs regenerated the degenerated NP and provide a simplified and efficient strategy for treating IVD degeneration.